Thiolalkyl benzoic acid derivatives

ABSTRACT

The present invention relates to new thiolalkyl benzoic acids, pharmaceutical compositions and diagnostic kits comprising such compounds, and methods of using such compounds for inhibiting NAALADase enzyme activity, detecting diseases where NAALADase levels are altered, effecting neuronal activity, effecting TGF-β activity, inhibiting angiogenesis, and treating glutamate abnormalities, neuropathy, pain, compulsive disorders, prostate diseases, cancers, glaucoma, retinal disorders, and cancer.

[0001] This application claims the benefit of U.S. Provisional PatentApplication Nos. 60/294,036 and 60/342,746 filed on May 30, 2001, andDec. 28, 2001, respectively, the entire disclosures of which areincorporated herein by reference.

[0002] The present invention relates to new compounds, pharmaceuticalcompositions and diagnostic kits comprising such compounds, and methodsof using such compounds for inhibiting NAALADase enzyme activity,detecting diseases where NAALADase levels are altered, effectingneuronal activity, effecting TGF-β activity, inhibiting angiogenesis,and treating glutamate abnormalities, neuropathy, pain, compulsivedisorders, prostate diseases, cancers, glaucoma, and retinal disorders.

[0003] The NAALADase enzyme, also known as prostate specific membraneantigen (“PSM” or “PSMA”) and human glutamate carboxypeptidase II (“GCPII”), catalyzes the hydrolysis of the neuropeptideN-acetyl-aspartyl-glutamate (“NAAG”) to N-acetyl-aspartate (“NAA”) andglutamate. Based upon amino acid sequence homology, NAALADase has beenassigned to the M28 family of peptidases.

[0004] Studies suggest NAALADase inhibitors may be effective in treatingischemia, spinal cord injury, demyelinating diseases, Parkinson'sdisease, Amyotrophic Lateral Sclerosis (“ALS”), alcohol dependence,nicotine dependence, cocaine dependence, cancer, neuropathy, pain andschizophrenia, and in inhibiting angiogenesis. In view of their broadrange of potential applications, a need exists for new NAALADaseinhibitors and pharmaceutical compositions comprising such compounds.

SUMMARY OF THE INVENTION

[0005] The present invention relates to a compound of formula Ia or apharmaceutically acceptable equivalent:

[0006] wherein:

[0007] R¹, R², R³, and R⁴ are independently hydrogen or C₁-C₃ alkyl; and

[0008] A¹, A², A³, and A⁴ are independently hydrogen, C₁-C₉ alkyl, C₂-C₉alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle, heterocycle, halo,hydroxy, sulfhydryl, nitro, amino, cyano, isocyano, thiocyano,isothiocyano, formamido, thioformamido, sulfo, sulfino, C₁-C₉alkylsulfonyl, C₁-C₉ alkoxy, C₂-C₉ alkenoxy, phenoxy, or benzyloxy,

[0009] wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl,carbocycle, heterocycle, alkoxy, alkenoxy, phenoxy, and benzyloxy areindependently unsubstituted or substituted with one or moresubstituent(s).

[0010] In one embodiment, R¹, R², R³, R⁴, A², A³, and A⁴ are hydrogen;and A¹ is hydrogen, —(CH₂)_(n)—W, or —Y—(CH₂)_(n)—W, wherein: n is 0-3;Y is O, S, or NR wherein R is hydrogen or C1-C4 alkyl; and W is C1-C6alkyl or phenyl, wherein W is unsubstituted or substituted with C1-C4alkyl, C1-C4 alkoxy, carboxy, or halo.

[0011] The present invention further relates to a compound of formula Ibor a pharmaceutically acceptable equivalent:

[0012] wherein A, A², A³ and A⁴ are independently hydrogen, C₁-C₉ alkyl,C₂-C₉ alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle, heterocycle,halo, hydroxy, sulfhydryl, nitro, amino, cyano, isocyano, thiocyano,isothiocyano, formamido, thioformamido, sulfo, sulfino, C₁-C₉alkylsulfonyl, C₁-C₉ alkoxy, C₂-C₉ alkenoxy, phenoxy, or benzyloxy,

[0013] wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl,carbocycle, heterocycle, alkoxy, alkenoxy, phenoxy, and benzyloxy areindependently unsubstituted or substituted with one or moresubstituent(s),

[0014] wherein if A¹ is chloro, fluoro, amino, or thiomethyl then A²,A³, and A⁴ may not all be hydrogen,

[0015] and wherein at least one of A¹, A², A³, and A⁴ is not hydrogen.

[0016] In one embodiment, A², A³, and A4 are hydrogen; and A¹ is—(CH₂)_(n)—Ar or —Y—(CH₂)_(n)—Ar, wherein n is 0-3, Y is O, S, or NRwherein R is hydrogen or C1-C4 alkyl, and Ar is phenyl, unsubstituted orsubstituted with C1-C4 alkyl, carboxy, or halo.

[0017] The present invention further relates to a compound of formula I:

[0018] or a pharmaceutically acceptable equivalent, wherein:

[0019] X is —(CR¹R²)_(n)SH, —O(CR¹R²)₂SH, —S(CR¹R²)₂SH, or—NR(CR¹R²)₂SH;

[0020] n is 1-3; and

[0021] R, R¹, R², A¹, A², A³ and A⁴ are independently hydrogen, C₁-C₉alkyl, C₂-C₉ alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle,heterocycle, halo, hydroxy, sulfhydryl, nitro, amino, cyano, isocyano,thiocyano, isothiocyano, formamido, thioformamido, sulfo, sulfino, C₁-C₉alkylsulfonyl, C₁-C₉ alkoxy, C₂-C₉ alkenoxy, phenoxy, or benzyloxy,

[0022] wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl,carbocycle, heterocycle, alkoxy, alkenoxy, phenoxy, and benzyloxy areindependently unsubstituted or substituted with one or moresubstituent(s).

[0023] Additionally, the present invention relates to a method forinhibiting NAALADase enzyme activity, detecting diseases where NAALADaselevels are altered, effecting neuronal activity, effecting TGF-β,activity, inhibiting angiogenesis, or treating glutamate abnormalities,neuropathy, pain, compulsive disorders, prostate diseases, cancers,glaucoma, or retinal disorders, comprising administering to a mammal inneed of such inhibition, treatment or effect, an effective amount of acompound of formula I, Ia, or Ib, as defined above.

[0024] The present invention further relates to a method for detecting adisease, disorder or condition where NAALADase levels are altered,comprising:

[0025] (i) contacting a sample of bodily tissue or fluid with a compoundof formula I, Ia, or Ib, as defined above, wherein said compound bindsto any NAALADase in said sample; and

[0026] (ii) measuring the amount of any NAALADase bound to said sample,wherein the amount of NAALADase is diagnostic for said disease,disorder, or condition.

[0027] The present invention also relates to a method for detecting adisease, disorder or condition where NAALADase levels are altered in ananimal or a mammal, comprising:

[0028] (i) labeling a compound of formula I, Ia, or Ib, as definedabove, with an imaging reagent;

[0029] (ii) administering to said animal or mammal an effective amountof the labeled compound;

[0030] (iii) allowing said labeled compound to localize and bind toNAALADase present in said animal or mammal; and

[0031] (iv) measuring the amount of NAALADase bound to said labeledcompound, wherein the amount of NAALADase is diagnostic for saiddisease, disorder, or condition.

[0032] Additionally, the present invention further relates to adiagnostic kit for detecting a disease, disorder, or condition whereNAALADase levels are altered, comprising a compound of formula I, Ia, orIb, as defined above, labeled with a marker.

[0033] Finally, the present invention relates to a pharmaceuticalcomposition comprising:

[0034] (i) an effective amount of a compound of formula I, Ia, or Ib, asdescribed above; and

[0035] (ii) a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1 is a bar graph showing the effect of Compound C on TGF-β1concentrations in ischernic cell cultures.

[0037]FIG. 2 is a bar graph showing the effect of Compound C on TGF-β2concentrations in ischemic cell cultures.

[0038]FIG. 3 is a bar graph showing the reversal of the neuroprotectiveeffect of Compound C by TGF-β neutralizing antibodies in ischemic cellcultures.

[0039]FIG. 4 is a bar graph showing the non-reversal of theneuroprotective effect of Compound C by FGF neutralizing antibodies inischemic cell cultures

[0040]FIG. 5 is a bar graph showing the reversal of the neuroprotectiveeffect of Compound C by TGF-β neutralizing antibodies in rats subjectedto middle cerebral artery occlusion (“MCAO”).

[0041]FIG. 6 is a bar graph showing the effect of Compound C on TGF-β1levels during occlusion and reperfusion in rats subjected to MCAO.

[0042]FIG. 7A is a bar graph plotting the withdrawal latency differencescores of non-diabetic rats and STZ-diabetic rats treated with a vehicleor Compound A, against the days following administration with STZ.

[0043]FIG. 7B is a bar graph plotting the withdrawal latency differencescores of non-diabetic rats and STZ-diabetic rats treated with a vehicleor Compound D, against the days following administration with STZ.

[0044]FIG. 8 is a bar graph plotting the withdrawal latency differencescores of normal (unoperated) rats and chronic constrictiveinjury-induced rats treated with a vehicle or Compound C, against thedays following surgery.

[0045]FIG. 9A is a bar graph plotting the motor nerve conductionvelocity of non-diabetic rats and STZ-diabetic rats treated with avehicle or Compound A, against the weeks following administration withSTZ.

[0046]FIG. 9B is a bar graph plotting the sensory nerve conductionvelocity of non-diabetic rats and STZ-diabetic rats treated with avehicle or Compound A, against the weeks following administration withSTZ.

[0047]FIG. 10A is a bar graph plotting the motor nerve conductionvelocity of non-diabetic rats and STZ-diabetic rats treated with avehicle or Compound D, against the weeks following administration withSTZ.

[0048]FIG. 10B is a bar graph plotting the sensory nerve conductionvelocity of non-diabetic rats and STZ-diabetic rats treated with avehicle or Compound D, against the weeks following administration withSTZ.

[0049]FIG. 11 is a graph plotting the withdrawal latency of non-diabeticrats and BB/W diabetic rats treated with a vehicle, Compound D, orCompound A, against the weeks of treatment.

[0050]FIG. 12 is a graph plotting the nerve conduction velocity ofnon-diabetic rats and BB/W diabetic rats treated with a vehicle,Compound D, or Compound A, against the weeks of treatment.

[0051]FIG. 13 is a bar graph plotting the withdrawal latency differencescores of chronic constrictive injury-induced rats treated with avehicle or varying amounts of Compound 9 against the days of treatment.

[0052]FIG. 14 is a bar graph plotting the withdrawal latency differencescores of chronic constrictive injury-induced rats treated with avehicle or Compound 10, against the days of treatment.

[0053]FIG. 15 is a bar graph plotting the percent of transgenic mice at210 days of age that exhibited limb shaking after treatment withCompound B or a vehicle.

[0054]FIG. 16 is a bar graph plotting the gait, measured on an arbitraryscale ranging from 0 to 3, of transgenic mice at 210 days of age aftertreatment with Compound B or a vehicle.

[0055]FIG. 17 is a bar graph plotting hind limbs dragging, measured onan arbitrary scale ranging from 0 to 3, of transgenic mice at 210 daysof age after treatment with Compound B or a vehicle.

[0056]FIG. 18 is a bar graph plotting the crossing of limbs, measured onan arbitrary scale ranging from 0 to 3, of transgenic mice at 210 daysof age after treatment with Compound B or a vehicle.

[0057]FIG. 19 is a bar graph plotting the righting reflex of transgenicmice, measured by the time (seconds) it took the mice to rightthemselves when placed on their sides, at 210 days of age aftertreatment with Compound B or a vehicle.

[0058]FIG. 20 is a graph plotting the percent of transgenic mice treatedwith Compound B or a vehicle that died against the age of the mice(days).

[0059]FIG. 21 is a Kaplan-Meier survival graph plotting the percent oftransgenic mice treated with Compound B or a vehicle that survivedagainst the number of days that the mice were on study therapy.

[0060]FIG. 22 shows the effect of treatment with Compounds D and E onneuropathic pain abnormalities in STZ diabetic rats.

[0061]FIG. 23 shows motor nerve conduction velocity measurements in STZdiabetic rats and non-diabetic controls prior to and after treatmentwith Compounds D and E.

[0062]FIG. 24 depicts sensory nerve conduction velocity deficits aftertreatment with Compounds D and E.

[0063]FIG. 25 shows neuropathic pain abnormalities with lower doses (1and 3 mg/kg) of Compound D initiated after 7 weeks of STZ treatment.

[0064]FIGS. 26 and 27 show sensory and motor nerve conduction velocityrespectively in chronically diabetic STZ rats treated with lower dosesof Compound D.

[0065]FIGS. 28 and 29 show sensory and motor nerve conduction velocitymeasurements where rats were left untreated until 60 days after STZtreatment.

[0066]FIG. 30 shows sensory nerve conduction velocity where treatmentwas delayed until 90 days after STZ.

[0067]FIG. 31 shows nerve conduction velocity measurements from agenetic mouse model of diabetes, at 6-7 months of age.

[0068]FIG. 32 shows nerve conduction velocity after 8 weeks of treatmentwith Compound F administered at 1 mg/kg daily.

[0069]FIG. 33 is bar graph comparing the rotarod performance oftransgenic HD mice and normal non-HD mice treated with Compound B, andtransgenic HD mice and normal non-HD mice treated with a vehicle.

[0070]FIG. 34 is a bar graph comparing the total distance traveled bytransgenic HD mice and normal non-HD mice treated with Compound B, andtransgenic HD mice and normal non-HD mice treated with a vehicle.

[0071]FIG. 35 is a graph plotting the survival time of transgenic D micetreated with Compound B or a vehicle.

[0072]FIG. 36 is a graph plotting the survival time of male transgenicHD mice treated with Compound B or a vehicle.

[0073]FIG. 37 is a graph plotting the survival time of female transgenicHD mice treated with Compound B or a vehicle.

DETAILED DESCRIPTION Definitions

[0074] “Compound A” refers to2-[[2,3,4,5,6-pentafluorobenzyl)hydroxyphosphinyl]methyl]pentanedioicacid.

[0075] “Compound B” refers to 2-(3-sulfanylpropyl)pentanedioic acid.

[0076] “Compound C” refers to 2-(phosphonomethyl)pentanedioic acid(“PMPA”).

[0077] “Compound D” refers to 2-(2-sulfanylethyl)pentanedioic acid.

[0078] “Compound E” refers to3-carboxy-alpha-(3-mercaptopropyl)-benzenepropanoic acid.

[0079] “Compound F” refers to3-carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)-benzenepropanoicacid.

[0080] “Compound 9” refers to2-[(4-carboxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid.

[0081] “Compound 10” refers to3-(2-mercaptoethyl)-[1,1′-biphenyl]-2,3′-dicarboxylic acid.

[0082] “Alkyl” refers to a branched or unbranched saturated hydrocarbonchain comprising a designated number of carbon atoms. For example, C₁-C₉alkyl is a straight or branched hydrocarbon chain containing 1 to 9carbon atoms, and includes but is not limited to substituents such asmethyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl,n-pentyl, n-hexyl, and the like, unless otherwise indicated.

[0083] “Alkenyl” refers to a branched or unbranched unsaturatedhydrocarbon chain comprising a designated number of carbon atoms. Forexample, C₂-C₉ alkenyl is a straight or branched hydrocarbon chaincontaining 2 to 9 carbon atoms having at least one double bond, andincludes but is not limited to substituents such as ethenyl, propenyl,iso-propenyl, butenyl, iso-butenyl, tert-butenyl, n-pentenyl, n-hexenyl,and the like, unless otherwise indicated.

[0084] “Alkoxy” refers to the group —OR wherein R is alkyl as hereindefined. Preferably, R is a branched or unbranched saturated hydrocarbonchain containing 1 to 9 carbon atoms.

[0085] “Carbocycle” refers to a hydrocarbon, cyclic moiety having one ormore closed ring(s) that is/are alicyclic, aromatic, fused, and/orbridged. Examples include cyclopropane, cyclobutane, cyclopentane,cyclohexane, cycloheptane, cyclopentene, cyclohexene, cycloheptene,cycloctene, benzyl, naphthene, anthracene, phenanthracene, biphenyl, andpyrene.

[0086] “Aryl” refers to an aromatic, hydrocarbon cyclic moiety havingone or more closed rings. Examples include, without limitation, phenyl,benzyl, naphthyl, anthracenyl, phenanthracenyl, biphenyl, and pyrenyl.

[0087] “Heterocycle” refers to a cyclic moiety having one or more closedrings that is/are alicyclic, aromatic, fused, and/or bridged, with oneor more heteroatoms (for example, sulfur, nitrogen or oxygen) in atleast one of the rings. Examples include, without limitation,pyrrolidine, pyrrole, thiazole, thiophene, piperidine, pyridine,isoxazolidine, and isoxazole.

[0088] “Heteroaryl” refers to an aromatic, cyclic moiety having one ormore closed rings with one or more heteroatoms (for example, sulfur,nitrogen, or oxygen) in at least one of the rings. Examples include,without limitation, pyrrole, thiophene, pyridine, and isoxazole.

[0089] “Derivative” refers to a substance produced from anothersubstance either directly or by modification or partial substitution.

[0090] “Effective amount” refers to the amount required to produce thedesired effect. “Therapeutically effective amount” refers to the amountrequired to inhibit NAALADase enzyme activity and/or angiogenesis, toeffect neuronal activity or TGF-β activity, and/or to treat glutamateabnormality, neuropathy, pain, compulsive disorder, prostate disease,cancer, glaucoma, and/or retinal disorders.

[0091] “Electromagnetic radiation” includes without limitation radiationhaving the wavelength of 10⁻²⁰ to 10⁰ meters. Examples include, withoutlimitation, gamma radiation (10⁻²⁰ to 10⁻¹³ m), X-ray radiation (10⁻¹¹to 10⁻⁹ m), ultraviolet light (10 nm to 400 nm), visible light (400 nmto 700 nm), infrared radiation (700 nm to 1.0 mm) and microwaveradiation (1 mm to 30 cm).

[0092] “Halo” refers to at least one fluoro, chloro, bromo, or iodomoiety.

[0093] “Isosteres” refer to elements, functional groups, substitutents,molecules or ions having different molecular formulae but exhibitingsimilar or identical physical properties. For example, tetrazole is anisostere of carboxylic acid because it mimics the properties ofcarboxylic acid even though they both have different molecular formulae.Typically, two isosteric molecules have similar or identical volumes andshapes. Ideally, isosteric compounds should be isomorphic and able toco-crystallize. Other physical properties that isosteric compoundsusually share include boiling point, density, viscosity and thermalconductivity. However, certain properties are usually different: dipolarmoments, polarity, polarization, size and shape since the externalorbitals may be hybridized differently. The term “isosteres” encompasses“bioisosteres.”

[0094] “Bioisosteres” are isosteres that, in addition to their physicalsimilarities, share some common biological properties. Typically,bioisosteres interact with the same recognition site or produce broadlysimilar biological effects.

[0095] “Carboxylic acid isosteres” include without limitation directderivatives such as hydroxamic acids, acyl-cyanamides, andacylsulfonamides; planar acidic heterocycles such as tetrazoles,mercaptoazoles, sulfinylazoles, sulfonylazoles, isoxazoles,isothiazoles, hydroxythiadiazoles, and hydroxychromes; and nonplanarsulfur- or phosphorus-derived acidic functions such as phosphinates,phosphonates, phosphonamides, sulphonates, sulphonamides, andacylsulphonamides.

[0096] “Metabolite” refers to a substance produced by metabolism or by ametabolic process.

[0097] “NAAG” refers to N-acetyl-aspartyl-glutamate, an importantpeptide component of the brain, with levels comparable to the majorinhibitor neurotransmitter gamma-aminobutyric acid (“GABA”). NAAG isneuron-specific, present in synaptic vesicles and released upon neuronalstimulation in several systems presumed to be glutamatergic. Studiessuggest that NAAG may function as a neurotransmitter and/orneuromodulator in the central nervous system, or as a precursor of theneurotransmitter glutamate. In addition, NAAG is an agonist at group IImetabotropic glutamate receptors, specifically mGluR3 receptors; whenattached to a moiety capable of inhibiting NAALADase, it is expectedthat metabotropic glutamate receptor ligands will provide potent andspecific NAALADase inhibitors.

[0098] “NAALADase” refers to N-acetylated α-linked acidic dipeptidase, amembrane bound metallopeptidase which catabolizes NAAG toN-acetylaspartate (“NAA”) and glutamate (“GLU”):

[0099] NAALADase has been assigned to the M28 peptidase family and isalso called PSMA or GCP II, EC number 3.4.17.21. It is believed thatNAALADase is a co-catalytic zinc/zinc metallopeptidase. NAALADase showsa high affinity for NAAG with a Km of 540 nM. If NAAG is a bioactivepeptide, then NAALADase may serve to inactivate NAAG'S synaptic action.Alternatively, if NAAG functions as a precursor for glutamate, theprimary function of NAALADase may be to regulate synaptic glutamateavailability.

[0100] “Pharmaceutically acceptable carrier” refers to any carrier,diluent, excipient, wetting agent, buffering agent, suspending agent,lubricating agent, adjuvant, vehicle, delivery system, emulsifier,disintegrant, absorbent, preservative, surfactant, colorant, flavorant,or sweetener, preferably non-toxic, that would be suitable for use in apharmaceutical composition.

[0101] “Pharmaceutically acceptable equivalent” includes, withoutlimitation, pharmaceutically acceptable salts, hydrates, metabolites,prodrugs, and isosteres. Many pharmaceutically acceptable equivalentsare expected to have the same or similar in vitro or in vivo activity asthe compounds of the invention.

[0102] “Pharmaceutically acceptable salt” refers to a salt of theinventive compounds which possesses the desired pharmacological activityand which is neither biologically nor otherwise undesirable. The saltcan be formed with acids that include, without limitation, acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfatebutyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloridehydrobromide, hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,thiocyanate, tosylate, and undecanoate. Examples of a base salt includeammonium salts, alkali metal salts such as sodium and potassium salts,alkaline earth metal salts such as calcium and magnesium salts, saltswith organic bases such as dicyclohexylamine salts,N-methyl-D-glucamine, and salts with amino acids such as arginine andlysine. Basic nitrogen-containing groups can be quarternized with agentsincluding lower alkyl halides such as methyl, ethyl, propyl and butylchlorides, bromides, and iodides; dialkyl sulfates such as dimethyl,diethyl, dibutyl, and diamyl sulfates; long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides, and iodides; andaralkyl halides such as benzyl and phenethyl bromides.

[0103] “Prodrug” refers to a derivative of the inventive compounds thatundergoes biotransformation, such as metabolism, before exhibiting itspharmacological effect(s). The prodrug is formulated with theobjective(s) of improved chemical stability, improved patient acceptanceand compliance, improved bioavailability, prolonged duration of action,improved organ selectivity, improved formulation (e.g., increasedhydrosolubility), and/or decreased side effects (e.g., toxicity). Theprodrug can be readily prepared from the inventive compounds usingmethods known in the art, such as those described by Burger's MedicinalChemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp. 172-178, 949-982(1995).

[0104] “Radiosensitizer” refers to a low molecular weight compoundadministered to animals in therapeutically effective amounts to promotethe treatment of diseases that are treatable with electromagneticradiation. Diseases that are treatable with electromagnetic radiationinclude, without limitation, neoplastic diseases, benign and malignanttumors, and cancerous cells. Electromagnetic radiation treatment ofother diseases not listed herein are also contemplated by the presentinvention.

[0105] “Inhibition,” in the context of enzymes, refers to reversibleenzyme inhibition such as competitive, uncompetitive and non-competitiveinhibition. Competitive, uncompetitive and non-competitive inhibitioncan be distinguished by the effects of an inhibitor on the reactionkinetics of an enzyme. Competitive inhibition occurs when the inhibitorcombines reversibly with the enzyme in such a way that it competes witha normal substrate for binding at the active site. The affinity betweenthe inhibitor and the enzyme may be measured by the inhibitor constant,K_(i), which is defined as:$K_{i} = \frac{\lbrack E\rbrack \lbrack I\rbrack}{\lbrack{EI}\rbrack}$

[0106] wherein [E] is the concentration of the enzyme, [I] is theconcentration of the inhibitor, and [EI] is the concentration of theenzyme-inhibitor complex formed by the reaction of the enzyme with theinhibitor. Unless otherwise specified, K_(i) as used herein refers tothe affinity between the inventive compounds and NAALADase. “IC₅₀” is arelated term used to define the concentration or amount of a compoundthat is required to cause a 50% inhibition of the target enzyme.

[0107] “NAALADase inhibitor” refers to any compound that inhibitsNAALADase enzyme activity. Preferably, a NAALADase inhibitor exhibits aK_(i) of less than 100 μM, more preferably less than 10 μM, and evenmore preferably less than 1 μM, as determined using any appropriateassay known in the art.

[0108] “Isomers” refer to compounds having the same number and kind ofatoms, and hence the same molecular weight, but differing in respect tothe arrangement or configuration of the atoms.

[0109] “Stereoisomers” are isomers that differ only in the arrangementof the atoms in space.

[0110] “Optical isomers” refer to enantiomers or diastereoisomers.

[0111] “Diastereoisomers” are stereoisomers that are not mirror imagesof each other. Diastereoisomers occur in compounds having two or moreasymmetric carbon atoms; thus, such compounds have 2^(n) opticalisomers, where n is the number of asymmetric carbon atoms.

[0112] “Enantiomers” are a pair of stereoisomers that arenon-superimposable mirror images of each other. Enantiomers result fromthe presence of one or more asymmetric carbon atoms in the compound(e.g., glyceraldehyde, lactic acid, sugars, tartaric acid, amino acids).

[0113] “Enantiomer-enriched” refers to a mixture in which one enantiomerpredominates.

[0114] “Racemic mixture” means a mixture containing equal amounts ofindividual enantiomers.

[0115] “Non-racemic mixture” is a mixture containing unequal amounts ofenantiomers.

[0116] “Angiogenesis” refers to the process whereby new capillaries areformed. “Inhibition” of angiogenesis may be measured by many parametersin accordance with the present invention and, for instance, may beassessed by delayed appearance of neovascular structures, sloweddevelopment of neovascular structures, decreased occurrence ofneovascular structures, slowed or decreased severity ofangiogenesis-dependent disease effects, arrested angiogenic growth, orregression of previous angiogenic growth. In the extreme, completeinhibition is referred to herein as prevention. In relation toangiogenesis or angiogenic growth, “prevention” refers to no substantialangiogenesis or angiogenic growth if none had previously occurred, or nosubstantial further angiogenesis or angiogenic growth if growth hadpreviously occurred.

[0117] “Angiogenesis-dependent disease” includes, without limitation,rheumatoid arthritis, cardiovascular diseases, neovascular diseases ofthe eye, peripheral vascular disorders, dermatologic ulcers, andcancerous tumor growth, invasion and metastasis.

[0118] “Animal” refers to a living organism having sensation and thepower of voluntary movement, and which requires for its existence oxygenand organic food. Examples include, without limitation, members of thehuman, equine, porcine, bovine, murine, canine, or feline species. Inthe case of a human, an “animal” may also be referred to as a “patient.”

[0119] “Mammal” refers to a warm-blooded vertebrate animal.

[0120] “Anxiety” includes without limitation the unpleasant emotionstate including psychophysiological responses to anticipation of unrealor imagined danger, ostensibly resulting from unrecognized intrapsychicconflict. Physiological concomitants include increased heart rate,altered respiration rate, sweating, trembling, weakness, and fatigue;psychological concomitants include feelings of impending danger,powerlessness, apprehension, and tension. Dorland's Illustrated MedicalDictionary, 27th ed. (W.B. Saunders Co. 1988).

[0121] “Anxiety Disorder” includes without limitation mental disordersin which anxiety and avoidance behavior predominate. Dorland'sIllustrated Medical Dictionary. Examples include without limitationpanic attack, agoraphobia, panic disorder, acute stress disorder,chronic stress disorder, specific phobia, simple phobia, social phobia,substance induced anxiety disorder, organic anxiety disorder, obsessivecompulsive disorder, post-traumatic stress disorder, generalized anxietydisorder, and anxiety disorder NOS. Other anxiety disorders arecharacterized in Diagnostic and Statistical Manual of Mental Disorders(American Psychiatric Association 4th ed. 1994).

[0122] “Attention Deficit Disorder” (“ADD”) refers to a disordercharacterized by developmentally inappropriate inattention andimpulsiveness, with or without hyperactivity. Inattention means afailure to finish tasks started, easily distracted, seeming lack ofattention, and difficulty concentrating on tasks requiring sustainedattention. Impulsiveness means acting before thinking, difficulty takingturns, problems organizing work, and constant shifting from one activityto another. Hyperactivity means difficulty staying seated and sittingstill, and running or climbing excessively.

[0123] “Cancer” includes, without limitation, ACTII-producing tumors,acute lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of theadrenal cortex, bladder cancer, brain cancer, breast cancer, cervixcancer, chronic lymphocytic leukemia, chronic myelocytic leukemia,colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cellleukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma,kidney cancer, liver cancer, lung cancer (small and/or non-small cell),malignant peritoneal effusion, malignant pleural effusion, melanoma,mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma,osteosarcoma, ovary cancer, ovary (germ cell) cancer, pancreatic cancer,penis cancer, prostate cancer, retinoblastoma, skin cancer, soft-tissuesarcoma, squamous cell carcinomas, stomach cancer, testicular cancer,thyroid cancer, trophoblastic neoplasms, cancer of the uterus, vaginalcancer, cancer of the vulva, and Wilm's tumor.

[0124] “Compulsive disorder” refers to any disorder characterized byirresistible impulsive behavior. Examples of compulsive disordersinclude without limitation substance dependence, eating disorders,pathological gambling, ADD, and Tourette's syndrome.

[0125] “Substance dependence” refers to a psychologic addiction or aphysical tolerance to a substance, e.g., a drug. Tolerance means a needto increase the dose progressively in order to produce the effectoriginally achieved by smaller amounts.

[0126] “Demyelinating disease” refers to any disease involving damage toor removal of the myelin sheath naturally surrounding nerve tissue, suchas that defined in U.S. Pat. No. 5,859,046 and International PublicationNo. WO 98/03178, herein incorporated by reference. Examples includewithout limitation peripheral demyelinating diseases (such asGuillain-Barré syndrome, peripheral neuropathies and Charcot-Marie Toothdisease) and central demyelinating diseases (such as multiplesclerosis).

[0127] “Disease” refers to any deviation from or interruption of thenormal structure or function of any part, organ or system (orcombinations) of the body that is manifested by a characteristic set ofsymptoms and signs and whose etiology, pathology, and prognosis may beknown or unknown. Dorland's Illustrated Medical Dictionary.

[0128] “Disorder” refers to any derangement or abnormality of function;a morbid physical or mental state. Dorland's Illustrated MedicalDictionary.

[0129] “Eating disorder” refers to compulsive overeating, obesity orsevere obesity. Obesity means body weight of 20% over standardheight-weight tables. Severe obesity means over 100% overweight.

[0130] “Glaucoma” includes without limitation chronic (idiopathic)open-angle glaucomas (e.g., high-pressure, normal-pressure); pupillaryblock glaucomas (e.g., acute angle-closure, subacute angle-closure,chronic angle-closure, combined-mechanism); developmental glaucomas(e.g., congenital (infantile), juvenile, Anxenfeld-Rieger syndrome,Peters' anomaly, Aniridia); glaucomas associated with other oculardisorders (e.g., glaucomas associated with disorders of the cornealendothelium, iris, ciliary body, lens, retina, choroid and vitreous);glaucomas associated with elevated episcleral venous pressure (e.g.,systemic diseases with associated elevated intraocular pressure andglaucoma, corticosteroid-induced glaucoma); glaucomas associated withinflammation and trauma (e.g., glaucomas associated with keratitis,episcleritis, scleritis, uveitis, ocular trauma, and hemorrhage);glaucomas following intraocular surgery, e.g., ciliary block (malignant)glaucoma, glaucomas in aphakia and pseudophakia, glaucomas associatedwith corneal surgery, glaucomas associated with vitreoretinal surgery.

[0131] “Glutamate abnormality” refers to any disease, disorder, orcondition in which glutamate is implicated, including pathologicalconditions involving elevated levels of glutamate. Examples of glutamateabnormalities include, without limitation, spinal cord injury, epilepsy,stroke, Alzheimer's disease, Parkinson's disease, ALS, Huntington'sdisease (“HD”), schizophrenia, pain, ischemia, peripheral neuropathy(including but not limited to neuropathy), traumatic brain injury,neuronal insult, inflammatory diseases, anxiety, anxiety disorders,memory impairment, compulsive disorders, glaucoma, and/or retinaldisorders.

[0132] “Ischemia” refers to localized tissue anemia due to obstructionof the inflow of arterial blood. Global ischemia occurs when blood flowceases for a period of time, as may result from cardiac arrest. Focalischemia occurs when a portion of the body, such as the brain, isdeprived of its normal blood supply, such as may result fromthromboembolytic occlusion of a cerebral vessel, traumatic head injury,edema or brain tumor. Even if transient, both global and focal ischemiacan produce widespread neuronal damage. Although nerve tissue damageoccurs over hours or even days following the onset of ischemia, somepermanent nerve tissue damage may develop in the initial minutesfollowing cessation of blood flow to the brain. Much of this damage isattributed to glutamate toxicity and secondary consequences ofreperfusion of the tissue, such as the release of vasoactive products bydamaged endothelium, and the release of cytotoxic products, such as freeradicals and leukotrienes, by the damaged tissue.

[0133] “Memory impairment” refers to a diminished mental registration,retention or recall of past experiences, knowledge, ideas, sensations,thoughts or impressions. Memory impairment may affect short andlong-term information retention, facility with spatial relationships,memory (rehearsal) strategies, and verbal retrieval and production.Common causes of memory impairment are age, severe head trauma, brainanoxia or ischemia, alcoholic-nutritional diseases, drug intoxicationsand neurodegenerative diseases. For example, memory impairment is acommon feature of neurodegenerative diseases such as Alzheimer's diseaseand senile dementia of the Alzheimer type. Memory impairment also occurswith other kinds of dementia such as multi-infarct dementia, a seniledementia caused by cerebrovascular deficiency, and the Lewy-body variantof Alzheimer's disease with or without association with Parkinson'sdisease. Creutzfeldt-Jakob disease is a rare dementia with which memoryimpairment is associated. It is a spongiform encephalopathy caused bythe prion protein; it may be transmitted from other sufferers or mayarise from gene mutations. Loss of memory is also a common feature ofbrain-damaged patients. Brain damage may occur, for example, after aclassical stroke or as a result of an anaesthetic accident, head trauma,hypoglycemia, carbon monoxide poisoning, lithium intoxication, vitamin(B₁, thiamine and B₁₂) deficiency, or excessive alcohol use. Korsakoff'samnesic psychosis is a rare disorder characterized by profound memoryloss and confabulation, whereby the patient invents stories to concealhis or her memory loss. It is frequently associated with excessivealcohol intake. Memory impairment may furthermore be age-associated; theability to recall information such as names, places and words seems todecrease with increasing age. Transient memory loss may also occur inpatients, suffering from a major depressive disorder, afterelectro-convulsive therapy.

[0134] “Mental disorder” refers to any clinically significant behavioralor psychological syndrome characterized by the presence of distressingsymptoms or significant impairment of functioning. Mental disorders areassumed to result from some psychological or organic dysfunction of theindividual; the concept does not include disturbances that areessentially conflicts between the individual and society (socialdeviance).

[0135] “Metastasis” refers to “[t]he ability of cells of a cancer todisseminate and form new foci of growth at noncontiguous sites (ie., toform metastases).” See Hill, R. P, “Metastasis”, The Basic Science ofOncology, Tannock et al., Eds., pp. 178-195 (McGraw-Hill 1992), hereinincorporated by reference. “The transition from in situ tumor growth tometastatic disease is defined by the ability of tumor cells of theprimary site to invade local tissues and to cross tissue barriers . . .To initiate the metastatic process, carcinoma cells must first penetratethe epithelial basement membrane and then invade the interstitial stroma. . . For distant metastases, intravasation requires tumor cell invasionof the subendothelial basement membrane that must also be negotiatedduring tumor cell extravasation . . . The development of malignancy isalso associated with tumor-induced angiogenesis [which] not only allowsfor expansion of the primary tumors, but also permits easy access to thevascular compartment due to defects in the basement membranes of newlyformed vessels.” See Aznavoorian et al., Cancer (1993) 71:1368-1383,herein incorporated by reference.

[0136] “Nervous insult” refers to any damage to nervous tissue and anydisability or death resulting therefrom. The cause of nervous insult maybe metabolic, toxic, neurotoxic, iatrogenic, thermal or chemical, andincludes without limitation ischemia, hypoxia, cerebrovascular accident,trauma, surgery, pressure, mass effect, hemorrhage, radiation,vasospasm, neurodegenerative disease, neurodegenerative process,infection, Parkinson's disease, ALS, myelination/demyelinationprocesses, epilepsy, cognitive disorder, glutamate abnormality andsecondary effects thereof.

[0137] “Nervous tissue” refers to the various components that make upthe nervous system, including without limitation neurons, neural supportcells, glia, Schwann cells, vasculature contained within and supplyingthese structures, the central nervous system, the brain, the brain stem,the spinal cord, the junction of the central nervous system with theperipheral nervous system, the peripheral nervous system and alliedstructures.

[0138] “Neuropathy” refers to any disease or malfunction of the nerves.Neuropathy includes, without limitation, peripheral neuropathy, diabeticneuropathy, autonomic neuropathy and mononeuropathy. Peripheralneuropathy may be idiopathic or induced by any causes including diseases(for example, amyloidosis, alcoholism, HIV, syphilis, virus, autoimmunedisorder, cancer, porphyria, arachnoiditis, post herpetic neuralgia,Guillain-Barré syndrome, diabetes including Type I and Type IIdiabetes), chemicals (for example, toxins, lead, dapsone, vitamins,paclitaxel chemotherapy, HAART therapy) and physical injuries to aparticular nerve or nerve plexus (for example, trauma, compression,constriction).

[0139] “Neuroprotective” refers to the effect of reducing, arresting orameliorating nervous insult, and protecting, resuscitating or revivingnervous tissue that has suffered nervous insult.

[0140] “Pain” refers to localized sensations of discomfort, distress oragony, resulting from the stimulation of specialized nerve endings. Itserves as a protective mechanism insofar as it induces the sufferer toremove or withdraw from the source. Dorland's Illustrated MedicalDictionary. Examples of pain include, without limitation, acute,chronic, cancer, burn, incisional, inflammatory, diabetic neuropathicand back pain.

[0141] “Neuropathic pain” refers to a condition of pain associated witha nerve injury. Depending on the particular syndrome, the pain may bedue to alterations of the brain or spinal cord or may be due toabnormalities in the nerve itself. Neuropathic pain may be idiopathic orinduced by any causes including diseases (for example, amyloidosis,alcoholism, HIV, syphilis, virus, autoimmune disorder, cancer,porphyria, arachnoiditis, post herpetic neuralgia, Guillain-Barrésyndrome, and diabetes, including Type I and Type II diabetes),chemicals (for example, toxins, lead, dapsone, vitamins, paclitaxelchemotherapy, and HAART therapy) and physical injuries to a particularnerve or nerve plexus (for example, trauma, compression, andconstriction).

[0142] “Pathological gambling” refers to a condition characterized by apreoccupation with gambling. Similar to psychoactive substance abuse,its effects include development of tolerance with a need to gambleprogressively larger amounts of money, withdrawal symptoms, andcontinued gambling despite severe negative effects on family andoccupation.

[0143] “Prostate disease” refers to any disease affecting the prostate.Examples of prostate disease include without limitation prostate cancersuch as adenocarcinoma and metastatic cancers of the prostate; andconditions characterized by abnormal growth of prostatic epithelialcells such as benign prostatic hyperplasia.

[0144] “Schizophrenia” refers to a mental disorder or group of mentaldisorders characterized by disturbances in form and content of thought(loosening of associations, delusions, hallucinations), mood (blunted,flattened, inappropriate affect), sense of self and relationship to theexternal world (loss of ego boundaries, dereistic thinking, and autisticwithdrawal), and behavior (bizarre, apparently purposeless, andstereotyped activity or inactivity). Examples of schizophrenia include,without limitation, acute, ambulatory, borderline, catatonic, childhood,disorganized, hebephrenic, latent, nuclear, paranoid, paraphrenic,prepsychotic, process, pseudoneurotic, pseudopsychopathic, reactive,residual, schizo-affective and undifferentiated schizophrenia. Dorland'sIllustrated Medical Dictionary.

[0145] “TGF-β” refers to transforming growth factor beta. TGF-β isrecognized as a prototype of multifunctional growth factors. Itregulates various cell and tissue functions, including cell growth anddifferentiation, angiogenesis, wound healing, immune function,extracellular matrix production, cell chemotaxis, apoptosis andhematopoiesis.

[0146] “TGF-β abnormality” refers to any disease, disorder or conditionin which TGF-β is implicated, including diseases disorders andconditions characterized by an abnormal level of TGF-β.

[0147] “Abnormal level of TGF-β” refers to a measurable variance fromnormal levels of TGF-β, as determined by one of ordinary skill in theart using known techniques.

[0148] “Therapeutic window of opportunity” or “window” refers, inrelation to stroke, to the maximal delay between the onset of stroke andthe initiation of efficacious therapy.

[0149] “Tourette's syndrome” refers to an autosomal multiple ticdisorder characterized by compulsive swearing, multiple muscle tics andloud noises. Tics are brief, rapid, involuntary movements that can besimple or complex; they are stereotyped and repetitive, but notrhythmic. Simple tics, such as eye blinking, often begin as nervousmannerisms. Complex tics often resemble fragments of normal behavior.

[0150] “Treating” refers to:

[0151] (i) preventing a disease, disorder or condition from occurring inan animal that may be predisposed to the disease, disorder and/orcondition but has not yet been diagnosed as having it;

[0152] (ii) inhibiting the disease, disorder or condition, i.e.,arresting its development; and/or

[0153] (iii) relieving the disease, disorder or condition, i.e., causingregression of the disease, disorder and/or condition.

[0154] “Treating ALS” refers to:

[0155] (i) preventing ALS from occurring in an animal that may bepredisposed to ALS but has not yet been diagnosed as having it;

[0156] (ii) inhibiting ALS, e.g., arresting its development;

[0157] (iii) relieving ALS, e.g., causing regression of the disease,disorder and/or condition;

[0158] (iv) delaying onset of ALS or ALS symptom(s);

[0159] (v) slowing progression of ALS or ALS symptom(s);

[0160] (vi) prolonging survival of an animal suffering from ALS; and/or

[0161] (vii) attenuating ALS symptom(s).

[0162] “Treating Huntington's disease” refers to:

[0163] (i) preventing Huntington's disease from occurring in an animalthat may be predisposed to Huntington's disease but has not yet beendiagnosed as having it;

[0164] (ii) inhibiting or slowing Huntington's disease, e.g. arrestingits development;

[0165] (iii) relieving Huntington's disease, e.g. causing itsregression;

[0166] (iv) improving motor coordination in an animal havingHuntington's disease; and/or

[0167] (v) prolonging the survival of an animal having Huntington'sdisease.

[0168] “Treating substance dependence” refers to suppressing thepsychologic addiction or physical tolerance to the drug of abuse, and/orrelieving and/or preventing a withdrawal syndrome resulting from thedrug dependence. “Dependence” refers to a maladaptive pattern ofsubstance use, leading to clinically significant impairment or distress.Dependence is typically characterized by tolerance and/or withdrawal.Substances for which dependence may be developed include, withoutlimitation, depressants (opioids, synthetic narcotics, barbiturates,glutethimide, methyprylon, ethchlorvynol, methaqualone, alcohol);anxiolytics (diazepam, chlordiazepoxide, alprazolam, oxazepam,temazepam); stimulants (amphetamine, methamphetamine, cocaine); andhallucinogens (LSD, mescaline, peyote, marijuana).

[0169] “Tolerance” refers to an acquired reaction to a substancecharacterized by diminished effect with continued use of the same doseand/or a need for increased doses to achieve intoxication or desiredeffect previously achieved by lower doses. Both physiological andpsychosocial factors may contribute to the development of tolerance.With respect to physiological tolerance, metabolic and/or functionaltolerance may develop. By increasing the rate of metabolism of thesubstance, the body may be able to eliminate the substance more readily.Functional tolerance is defined as a decrease in sensitivity of thecentral nervous system to the substance.

[0170] “Withdrawal” refers to a syndrome characterized by untowardphysical changes that occur following cessation of or reduction insubstance use, or administration of a pharmacologic antagonist.

[0171] “Treating a retinal disorder” refers to:

[0172] (i) preventing a retinal disorder from occurring in an animalthat may be predisposed to a retinal disorder but has not yet beendiagnosed as having it;

[0173] (ii) inhibiting a retinal disorder, e.g., arresting itsdevelopment; and/or

[0174] (iii) relieving a retinal disorder, e.g., causing its regression.

[0175] “Retinal disorder” refers to vascular retinopathy, for example,hypertensive retinopathy, diabetic retinopathy (nonproliferative orproliferative), central retinal artery occlusion, or central retinalvein occlusion; age-related macular degeneration; retinal detachment; orretinitis pigmentosa.

[0176] One of ordinary skill in the art will recognize that there arealternative nomenclatures, nosologies and classification systems for thediseases, disorders and conditions defined above, and that such systemsevolve with medical scientific progress.

[0177] Unless the context clearly dictates otherwise, the definitions ofsingular terms may be extrapolated to apply to their plural counterpartsas they appear in the application; likewise, the definitions of pluralterms may be extrapolated to apply to their singular counterparts asthey appear in the application.

COMPOUNDS OF THE INVENTION

[0178] The present invention relates to a compound of formula Ia:

[0179] wherein A¹, A², A³ and A⁴ are independently hydrogen, C₁-C₉alkyl, C₂-C₉ alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle,heterocycle, halo, hydroxy, sulfhydryl, nitro, amino, cyano, isocyano,thiocyano, isothiocyano, formamido, thioformarnido, sulfo, sulfino,C₁-C₉ alkylsulfonyl, C₁-C₉ alkoxy, C₂-C₉ alkenoxy, phenoxy, orbenzyloxy,

[0180] wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl,carbocycle, heterocycle, alkoxy, alkenoxy, phenoxy, and benzyloxy areindependently unsubstituted or substituted with one or moresubstituent(s).

[0181] In one embodiment, R¹, R², R³, R⁴, A², A³, and A⁴ are hydrogen;and A¹ is hydrogen, —(CH₂)_(n)—W, or —Y—(CH₂)_(n)—W, wherein: n is 0-3;Y is O, S, or NR wherein R is hydrogen or C1-C4 alkyl; and W is C1-C6alkyl or phenyl, wherein W is unsubstituted or substituted with C1-C4alkyl, C1-C4 alkoxy, carboxy, or halo.

[0182] The present invention further relates to a compound of formulaIb:

[0183] wherein A¹, A², A³ and A⁴ are independently hydrogen, C₁-C₉alkyl, C₂-C₉ alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle,heterocycle, halo, hydroxy, sulfhydryl, nitro, amino, cyano, isocyano,thiocyano, isothiocyano, formamido, thioformamido, sulfo, sulfino, C₁-C₉alkylsulfonyl, C₁-C₉ alkoxy, C₂-C₉ alkenoxy, phenoxy, or benzyloxy,

[0184] wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl,carbocycle, heterocycle, alkoxy, alkenoxy, phenoxy, and benzyloxy areindependently unsubstituted or substituted with one or moresubstituent(s),

[0185] wherein if A¹ is chloro, fluoro, amino, or thiomethyl then A²,A³, and A⁴ may not all be hydrogen,

[0186] and wherein at least one of A¹, A², A³, and A⁴ is not hydrogen.

[0187] In one embodiment, A², A³, and A⁴ are hydrogen; and A¹ is—(CH₂)_(n)—Ar or —Y—(CH₂)_(n)—Ar, wherein n is 0-3, Y is O, S, or NRwherein R is hydrogen or C1-C4 alkyl, and Ar is phenyl, unsubstituted orsubstituted with C1-C4 alkyl, carboxy, or halo.

[0188] The present invention further relates to a compound of formula I

[0189] or a pharmaceutically acceptable equivalent, wherein:

[0190] X is —(CR¹R²)_(n)SH, —O(CR¹R²)₂SH, —S(CR¹R²)₂SH, or—NR(CR¹R²)₂SH;

[0191] n is 1-3; and

[0192] R, R¹, R², A¹, A², A³ and A⁴ are independently hydrogen, C₁-C₉alkyl, C₂-C₉ alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle,heterocycle, halo, hydroxy, sulfhydryl, nitro, amino, cyano, isocyano,thiocyano, isothiocyano, formamido, thioformamido, sulfo, sulfino, C₁-C₉alkylsulfonyl, C₁-C₉ alkoxy, C₂-C₉ alkenoxy, phenoxy, or benzyloxy,

[0193] wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl,carbocycle, heterocycle, alkoxy, alkenoxy, phenoxy, and benzyloxy areindependently unsubstituted or substituted with one or moresubstituent(s).

[0194] Possible substituents of said alkyl, alkenyl, alkynyl, aryl,heteroaryl, carbocycle, heterocycle, alkoxy, alkenyloxy, phenoxy,benzyloxy, and fused ring include, without limitation, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, C₂-C₆ alkenyloxy, phenoxy,benzyloxy, hydroxy, carboxy, hydroperoxy, carbamido, carbamoyl,carbamyl, carbonyl, carbozoyl, amino, hydroxyamino, formamido, formyl,guanyl, cyano, cyanoamino, isocyano, isocyanato, diazo, azido,hydrazino, triazano, nitrilo, nitro, nitroso, isonitroso, nitrosamino,imino, nitrosimino, oxo, C₁-C₆ alkylthio, sulfamino, sulfamoyl, sulfeno,sulfhydryl, sulfinyl, sulfo, sulfonyl, thiocarboxy, thiocyano,isothiocyano, thioformamido, halo, haloalkyl, chlorosyl, chloryl,perchloryl, trifluoromethyl, iodosyl, iodyl, phosphino, phosphinyl,phospho, phosphono, arsino, selanyl, disilanyl, siloxy, silyl, silylene,and carbocyclic and heterocyclic moieties. Carbocyclic moieties includealicyclic and aromatic structures.

[0195] Examples of carbocyclic and heterocyclic moieties include,without limitation, phenyl, benzyl, naphthyl, indenyl, azulenyl,fluorenyl, anthracenyl, indolyl, isoindolyl, indolinyl, benzofuranyl,benzothiophenyl, indazolyl, benzimidazolyl, benzthiazolyl,tetrahydrofuranyl, tetrahydropyranyl, pyridyl, pyrrolyl, pyrrolidinyl,pyridinyl, pyrimidinyl, purinyl, quinolinyl, isoquinolinyl,tetrahydroquinolinyl, quinolizinyl, furyl, thiophenyl, imidazolyl,oxazolyl, benzoxazolyl, thiazolyl, isoxazolyl, isotriazolyl,oxadiazolyl, triazolyl, thiadiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, trithianyl, indolizinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, thienyl, tetrahydroisoquinolinyl, cinnolinyl,phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl,carbazolyl, acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl.

[0196] Representative compounds of the present invention are set forthbelow in Table I. TABLE I Compound No. Structure Name 1

2-(2-mercaptoethyl)-benzoic acid 2

5-hydroxy-2-(2-mercaptoethyl)-benzoic acid 3

5-[(4-carboxyphenyl)methoxy]-2-(2- mercaptoethyl)-benzoic acid 4

2-(2-mercaptoethyl)-5-(phenylmethoxy)-benzoic acid, 5

2-(carboxymethoxy)-6-(2-mercaptoethyl)- benzoic acid 6

5-[(3-carboxyphenyl)methoxy]-2-(2- mercaptoethyl)-benzoic acid 7

2-(2-mercaptoethyl)-6-(phenylmethoxy)- benzoic acid 8

2-[(2-carboxyphenyl)methoxy]-6-(2- mercaptoethyl)-benzoic acid 9

2-[(4-carboxyphenyl)methoxy]-6-(2- mercaptoethyl)-benzoic acid 10

3-(2-mercaptoethyl)-[1,1′-biphenyl]-2,3′- dicarboxylic acid 11

5-(mercaptomethyl)-2-(2-phenylethoxy)- benzoic acid 12

2-(3,3-dimethylbutoxy)-6-(2- mercaptoethyl)-benzoic acid 13

2-(2-mercaptoethyl)-6-(2-phenylethoxy)- benzoic acid 14

2-[(2-chlorophenyl)methoxyl]-6-(2- mercaptoethyl)-benzoic acid 15

2-[[3-carboxy-5-(1,1- dimethylethyl)phenyl]methoxy]-6-(2-mercaptoethyl)-benzoic acid 16

3-(2-mercaptoethyl)-[1,1′-biphenyl]-2,4′- dicarboxylic acid 17

2-[(4-carboxy-2- methoxyphenyl)methoxy]-6-(2- mercaptoethyl)-benzoicacid 18

2-[(4-carboxy-3- methoxyphenyl)methoxy]-6-(2- mercaptoethyl)-benzoicacid 19

2-[(2-bromo-4-carboxyphenyl)methoxy]- 6-(2-mercaptoethyl)-benzoic acid20

2-[(3-bromo-4-carboxyphenyl)methoxy]- 6-(2-mercaptoethyl)-benzoic acid21

2-(2-mercaptoethyl)-6-phenoxy-benzoic acid 22

2-(2-mercaptoethyl)-6-phenylamino-benzoic acid 23

2-(2-mercaptoethyl)-6-(phenylthio)-benzoic acid 24

5′-(1,1-dimethylethyl)-3-(2- mercaptoethyl)-[1,1′-biphenyl]-2,3′-dicarboxylic acid 25

2-bromo-5-(mercaptomethyl)-benzoic acid 26

4-(mercaptomethyl)-[1,1′-biphenyl]-2,3′- dicarboxylic acid 27

5-(mercaptomethyl)-2-(phenylmethoxy)- benzoic acid 28

4-bromo-3-(mercaptomethyl)-benzoic acid 29

3-(2-mercaptoethyl)-benzoic acid 30

3-(mercaptomethyl)-benzoic acid 31

2-(mercaptomethyl)-benzoic acid 32

2-[(4-chlorophenyl)methoxy]-6-(2- mercaptoethyl)-benzoic acid 33

2-(biphenyl-2-ylmethoxy)-6-(2- mercaptoethyl)-benzoic acid 34

2-[(3-bromo-5-carboxyphenyl) methoxy]- 6-(2-mercaptoethyl)-benzoic acid35

2-[(2-bromo-5-carboxyphenyl) methoxyl]- 6-(2-mercaptoethyl)-benzoic acid36

2-(2-mercaptoethyl)-6-[(4- methoxyphenyl)methoxy]-benzoic acid 37

2-(2-mercaptoethyl)-6-[(4- methylphenyl)methoxy]-benzoic acid 38

2-[(4-bromo-3-carboxyphenyl) methoxy]- 6-(2-mercaptoethyl)-benzoic acid39

2-[(2-carboxy-5-methoxyphenyl) methoxy]-6-(2-mercaptoethyl)- benzoicacid 40

2-(3-carboxy-benzyloxy)-6-(2-mercapto- ethyl)-benzoic acid 41

2-(4-bromo-benzyloxy)-6-(2-mercapto- ethyl)-benzoic acid 42

2-(4-tert-butyl-benzyloxy)-6-(2-mercapto- ethyl)-benzoic acid 43

2-(3-bromo-benzyloxy)-6-(2-mercapto- ethyl)-benzoic acid 44

2-(2-mercapto-ethyl)-6-methoxy-benzoic acid 45

2-benzhydryloxy-6-(2-mercapto-ethyl)- benzoic acid 46

2-(3-chloro-benzyloxy)-6-(2-mercapto- ethyl)-benzoic acid 47

3-(2-mercapto-ethyl)-biphenyl-2-carboxylic acid 48

2-carboxymethyl-6-(2-mercapto-ethyl)- benzoic acid

[0197] The compounds of the invention possess one or more asymmetriccarbon center(s) and are thus capable of existing in the form of opticalisomers as well as in the form of racemic or non-racemic mixtures ofoptical isomers. The optical isomers can be obtained by resolution ofthe racemic mixtures according to conventional processes well known inthe art, for example by formation of diastereoisomeric salts bytreatment with an optically active acid or base and then separation ofthe mixture of diastereoisomers by crystallization followed byliberation of the optically active bases from these salts. Examples ofuseful acids include tartaric, diacetyltartaric, dibenzoyltartaric,ditoluoyltartaric, and camphorsulfonic acids.

[0198] A different process for separating optical isomers involves theuse of a chiral chromatography column optimally chosen to maximize theseparation of the enantiomers. Still another available method involvessynthesis of covalent diastereoisomeric molecules, for example, esters,amides, acetals, ketals, and the like, by reacting compounds used in theinventive methods and pharmaceutical compositions with an opticallyactive acid in an activated form, an optically active diol or anoptically active isocyanate. The synthesized diastereoisomers can beseparated by conventional means, such as chromatography, distillation,crystallization or sublimation, and then hydrolyzed to deliver theenantiomerically pure compound. In some cases, hydrolysis to the parentoptically active drug prior to dosing the patient is unnecessary sincethe compound can behave as a prodrug. The optically active compounds ofthe present invention can likewise be obtained by utilizing opticallyactive starting materials.

[0199] It is understood that the compounds of the invention encompassoptical isomers as well as racemic and non-racemic mixtures.

METHODS OF THE INVENTION Methods for Inhibiting NAALADase EnzymeActivity

[0200] The present invention relates to a method for inhibitingNAALADase enzyme activity in an animal or a mammal, comprisingadministering to said animal or mammal an effective amount of a compoundof the invention, as defined above.

Methods for Treating Glutamate Abnormalities

[0201] The present invention further relates to a method for treating aglutamate abnormality in an animal or a mammal, comprising administeringto said animal or mammal an effective amount of a compound of theinvention, as defined above.

[0202] Glutamate abnormalities to be treated may include compulsivedisorder, stroke, demyelinating disease, schizophrenia, Parkinson'sdisease, ALS, diabetic neuropathy, pain, anxiety, anxiety disorder,memory impairment, and glaucoma. Preferably, the compulsive disorder isalcohol, nicotine or cocaine dependence.

[0203] Stroke patients often experience a significant temporal delaybetween the onset of ischemia and the initiation of therapy. Thus, thereis a need for neuroprotectants with a long therapeutic window ofopportunity. It is expected that the compounds of the invention have atherapeutic window of opportunity of at least 1 hour. Accordingly, whenthe glutamate abnormality is stroke, the compound of the invention maybe administered to said animal or mammal for up to 60 minutes, 120minutes or more following onset of stroke.

[0204] Without being bound to any particular mechanism of action,preferred compounds of the present invention are expected to be thosethat block glutamate release pre-synaptically without interacting withpost-synaptic glutamate receptors. Such compounds would be devoid of thebehavioral toxicities associated with post-synaptic glutamateantagonists.

Methods for Effecting Neuronal Activities

[0205] The present invention further relates to a method for effecting aneuronal activity in an animal or a mammal, comprising administering tosaid animal or mammal an effective amount of a compound of theinvention, as defined above.

[0206] The neuronal activity that is effected by the inventive methodmay be stimulation of damaged neurons, promotion of neuronalregeneration, prevention of neurodegeneration or treatment of aneurological disorder.

[0207] Preferably, the neuronal activity is treatment of a neurologicaldisorder that is pain, neuropathy, traumatic brain injury, physicaldamage to spinal cord, stroke associated with brain damage, ademyelinating disease, or a neurological disorder relating toneurodegeneration.

[0208] Examples of neurological disorders that are treatable by themethods of the present invention include without limitation: trigeminalneuralgia; glossopharyngeal neuralgia; Bell's Palsy; myasthenia gravis;muscular dystrophy; ALS; progressive muscular atrophy; progressivebulbar inherited muscular atrophy; herniated, ruptured or prolapsedinvertebrate disk syndromes; cervical spondylosis; plexus disorders;thoracic outlet destruction syndromes; peripheral neuropathies such asthose caused by lead, dapsone, ticks, porphyria, or Guillain-Barrésyndrome; diabetic neuropathy; pain; Alzheimer's disease; andParkinson's disease.

[0209] The inventive method is particularly useful for treating aneurological disorder selected from the group consisting of peripheralneuropathy caused by physical injury or disease state, diabeticneuropathy, HIV-, chemical-, and vitamin-induced neuropathies, pain,traumatic brain injury, physical damage to spinal cord, strokeassociated with brain damage, demyelinating disease and neurologicaldisorder relating to neurodegeneration.

[0210] When the neurological disorder is pain, the compound of theinvention is preferably administered in combination with an effectiveamount of morphine.

[0211] The inventive method is particularly useful for treatingneuropathic pain, e.g., HIV-, chemical-, and vitamin-induced neuropathicpain.

[0212] Examples of neurological disorders relating to neurodegenerationinclude Alzheimer's disease, Parkinson's disease, and ALS.

Methods for Treating Prostrate Diseases

[0213] The present invention further relates to a method for treating aprostate disease in an animal or a mammal, comprising administering tosaid animal or mammal an effective amount of a compound of theinvention, as defined above. A preferred prostate disease is prostatecancer.

Methods for Treating Cancers

[0214] The present invention further relates to a method for treatingcancer in an animal or a mammal, comprising administering to said animalor mammal an effective amount of a compound of the invention, as definedabove.

[0215] Preferred cancers to be treated are those in tissues whereNAALADase resides, including without limitation the brain, kidney andtestis.

Methods for Inhibiting Angiogenesis

[0216] The present invention further relates to a method for inhibitingangiogenesis in an animal or a mammal, comprising administering to saidanimal or mammal an effective amount of a compound of the invention, asdefined above.

[0217] Angiogenesis may be necessary for fertility or metastasis ofcancer tumors, or may be related to an angiogenic-dependent disease.Thus, the inventive methods may also be useful for treating anangiogenic-dependent disease including, without limitation, rheumatoidarthritis, cardiovascular diseases, neovascular diseases of the eye,peripheral vascular disorders, dermatologic ulcers and cancerous tumorgrowth, invasion, or metastasis.

Methods for Effecting TGF-β Activity

[0218] The present invention further relates to a method for effecting aTGF-β activity in an animal or a mammal, comprising administering tosaid animal or mammal an effective amount of a compound of theinvention, as defined above.

[0219] Effecting a TGF-β activity includes increasing, reducing orregulating TGF-β levels, and treating TGF-β abnormalities. Examples ofTGF-β abnormalities to be treated include neurodegenerative disorders,extra-cellular matrix formation disorders, cell-growth related diseases,infectious diseases, immune related diseases, epithelial tissuescarring, collagen vascular diseases, fibroproliferative disorders,connective tissue disorders, inflammation, inflammatory diseases,respiratory distress syndrome, infertility and diabetes.

[0220] Typical neurodegenerative disorders to be treated include neuraltissue damage resulting from ischemia reperfusion injury, myelinationand neurodegeneration.

[0221] Typical cell-growth related disorders to be treated include thoseaffecting kidney cells, hematopoietic cells, lymphocytes, epithelialcells and endothelial cells.

[0222] Typical infectious diseases to be treated include those caused bya macrophage pathogen, particularly a macrophage pathogen selected fromthe group consisting of bacteria, yeast, fungi, viruses, protozoa,Trypanosoma cruzi, Histoplasma capsulatum, Candida albicans, Candidaparapsilosis, Cryptococcus neoformans, Salmonella, Pneumocystis,Toxoplasma, Listeria, Mycobacteria, Rickettsia and Leishmania.Mycobacteria include without limitation Mycobacterium tuberculosis andMycobacterium leprae. Toxoplasma includes without limitation Toxoplasmagondii. Rickettsia includes without limitation R. prowazekii, R.coronii, and R. tsutsugamushi.

[0223] Other examples of infectious diseases to be treated includesingle or multiple cutaneous lesions, mucosal disease, Chagas' disease,acquired immunodeficiency syndrome (“AIDS”), toxoplasmosis,leishmaniasis, trypanosomiasis, shistosomiasis, cryptosporidiosis,Mycobacterium avium infections, Pneumocystis carinii pneumonia andleprosy.

[0224] Typical immune related diseases to be treated include autoimmunedisorders; impaired immune function; and immunosuppression associatedwith an infectious disease, particularly, trypanosomal infection, viralinfection, human immunosuppression virus, human T cell lymphotropicvirus (“HTLV-1”), lymphocytic choriomeningitis virus, or hepatitis.

[0225] Typical collagen vascular diseases to be treated includeprogressive systemic sclerosis (“PSS”), polymyositis, scleroderma,dermatomyositis, eosinophilic fascitis, morphea, Raynaud's syndrome,interstitial pulmonary fibrosis, scleroderma and systemic lupuserythematosus.

[0226] Typical fibroproliferative disorders to be treated includediabetic nephropathy, kidney disease, proliferative vitreoretinopathy,liver cirrhosis, biliary fibrosis, and myelofibrosis. Especiallypreferred kidney diseases include mesangial proliferativeglomerulonephritis, crescentic glomerulonephritis, diabetic neuropathy,renal interstitial fibrosis, renal fibrosis in transplant patientsreceiving cyclosporin, and HIV-associated nephropathy.

[0227] Typical connective tissue disorders to be treated includescleroderma, myelofibrosis, and hepatic, intraocular and pulmonaryfibrosis.

[0228] Typical inflammatory diseases to be treated are associated withPSS, polymyositis, scleroderma, dermatomyositis, eosinophilic fascitis,morphea, Raynaud's syndrome, interstitial pulmonary fibrosis,scleroderma, systemic lupus erythematosus, diabetic nephropathy, kidneydisease, proliferative vitreoretinopathy, liver cirrhosis, biliaryfibrosis, myelofibrosis, mesangial proliferative glomerulonephritis,crescentic glomerulonephritis, diabetic neuropathy, renal interstitialfibrosis, renal fibrosis in transplant patients receiving cyclosporin,or HIV-associated nephropathy.

[0229] Without being limited to any particular mechanism of action,preferred compounds of the present invention treat inflammatory diseasesby regulating TGF-β and/or inhibiting myeloperoxidase.

[0230] Other uses associated with the inventive compounds' TGF-βregulating properties include:

[0231] stimulating growth of tissue, glands or organs, particularlygrowth that would enhance milk production or weight gain;

[0232] stimulating cell proliferation, particularly proliferation offibroblasts, mesenchymal cells or epithelial cells;

[0233] inhibiting cell growth, particularly of epithelial cells,endothelial cells, T and B lymphocytes and thymocytes;

[0234] inhibiting expression of adipose, skeletal muscle andhematopoietic phenotypes, neoplasms, non-cytocidal viral or otherpathogenic infections and autoimmune disorders;

[0235] mediating disease resistance and susceptibility;

[0236] suppressing cellular immune response;

[0237] inhibiting scar tissue formation, preferably in skin or otherepithelial tissue that has been damaged by wounds resulting fromaccidental injury, surgical operations, trauma-induced lacerations orother trauma, or wounds involving the peritoneum for which the excessiveconnective tissue formation is abdominal adhesions;

[0238] increasing the effectiveness of a vaccine, particularly a vaccinefor an allergy towards, for example, dust or hayfever; and

[0239] inhibiting polyp formation.

Methods for Treating a Retinal Disorder

[0240] The present invention further relates to a method of treating aretinal disorder comprising administering an effective amount of aNAALADase inhibitor to an animal in need of such treatment.

Diagnostic Methods and Kits

[0241] The compounds of the present invention are useful for in vitroand in vivo diagnostic methods for detecting diseases, disorders andconditions where NAALADase levels are altered including, withoutlimitation, neurological disorders, glutamate abnormalities, neuropathy,pain, compulsive disorders, prostate diseases, cancers, TGF-βabnormalities, and glaucoma.

[0242] Accordingly, the present invention also relates to a method fordetecting a disease, disorder or condition where NAALADase levels arealtered, comprising:

[0243] (i) contacting a sample of bodily tissue or fluid with a compoundof the invention, as defined above, wherein said compound binds to anyNAALADase in said sample; and

[0244] (ii) measuring the amount of any NAALADase bound to said sample,wherein the amount of NAALADase is diagnostic for said disease, disorderor condition.

[0245] Examples of bodily tissues and fluids include, withoutlimitation, prostate tissue, ejaculate, seminal vesicle fluid, prostaticfluid, urine, blood, saliva, tears, sweat, lymph and sputum.

[0246] The compound may be labeled with a marker using techniques knownin the art. Useful markers include, without limitation, enzymaticmarkers and imaging reagents. Examples of imaging reagents includeradiolabels such as ¹³¹I, ¹¹¹In, ¹²³I, ⁹⁹Tc, ³²P, ¹²⁵I, ³II, and ¹⁴C;fluorescent labels such as fluorescein and rhodamine; andchemiluminescers such as luciferin.

[0247] The amount of NAALADase can be measured using techniques known inthe art including, without limitation, assays (such as immunometric,calorimetric, densitometric, spectrographic and chromatographic assays)and imaging techniques (such as magnetic resonance spectroscopy (“MRS”),magnetic resonance imaging (“MRI”), single-photon emission computedtomography (“SPECT”), and positron emission tomography (“PET”)).

[0248] The present invention further relates to a diagnostic kit fordetecting a disease, disorder or condition where NAALADase levels arealtered. The diagnostic kit includes a compound of the invention, asdefined above, labeled with a marker. The diagnostic kit may furtherinclude buffering agents, agents for reducing background interference,control reagents and/or apparatus for conducting the test.

[0249] The present invention further relates to a method for detecting adisease, disorder or condition where NAALADase levels are altered in ananimal or a mammal, comprising:

[0250] (i) labeling a compound of the invention, as defined above, withan imaging reagent;

[0251] (ii) administering to said animal or mammal an effective amountof the labeled compound;

[0252] (iii) allowing said labeled compound to localize and bind toNAALADase present in said animal or mammal; and

[0253] (iv) measuring the amount of NAALADase bound to said labeledcompound, wherein the amount of NAALADase is diagnostic for saiddisease, disorder or condition.

[0254] The amount of NAALADase can be measured in vivo using knownimaging techniques, as described above.

INCORPORATION BY REFERENCE

[0255] The relationship between NAALADase inhibitors and glutamate, andthe effectiveness of NAALADase inhibitors in treating and detectingvarious diseases, disorders and conditions have been discussed in U.S.Pat. Nos. 5,672,592; 5,795,877; 5,804,602; 5,824,662; 5,863,536;5,977,090; 5,981,209; 6,011,021; 6,017,903; 6,025,344; 6,025,345;6,046,180; 6,228,888 and 6,265,609; International Publication Nos. WO00/01668 and WO 00/38785; provisional U.S. patent application Ser. No.60/261,754; and other references generally known in the art. The presentinventors hereby incorporate by reference, as though set forth herein infull, the entire contents of the aforementioned patents, patentapplications, and publications, particularly their discussions, figuresand data regarding the effectiveness of NAALADase inhibitors ininhibiting angiogenesis, in effecting TGF-β activity, in diagnosingdiseases, and in treating ischemia, spinal cord injury, demyelinatingdiseases, Parkinson's disease, ALS, alcohol dependence, nicotinedependence, cocaine dependence, prostate disease, cancer, neuropathy,pain, schizophrenia, anxiety, anxiety disorder, and memory impairment.The present inventors have discovered that the inventive compounds areeffective NAALADase inhibitors. Thus, the inventive compounds areexpected to have the same uses as the NAALADase inhibitors disclosed inthe patents, patent applications, and publications that are incorporatedby reference.

Pharmaceutical Compositions of the Invention

[0256] The present invention also relates to a pharmaceuticalcomposition comprising an effective amount of a compound of theinvention or a pharmaceutically acceptable equivalent; and apharmaceutically acceptable carrier.

[0257] Preferably, the compound of the invention is present in aneffective amount for inhibiting NAALADase enzyme activity orangiogenesis, effecting a neuronal activity or TGF-β activity, ortreating a glutamate abnormality, neuropathy, pain, prostate disease, orcancer in an animal or a mammal.

Route of Administration

[0258] In the inventive methods, the compounds will generally beadministered to a patient in the form of a pharmaceutical formulation.Such formulation preferably includes, in addition to the active agent, aphysiologically acceptable carrier and/or diluent. The compounds may beadministered locally or systemically by any means known to an ordinarilyskilled artisan. For example, the compounds may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir in dosage formulationscontaining conventional non-toxic pharmaceutically acceptable carriers,adjuvants and vehicles. The term parenteral as used herein includessubcutaneous, intravenous, intraarterial, intramuscular,intraperitoneal, intrathecal, intraventricular, intrastemal,intracranial or intraosseous injection and infusion techniques. Theexact administration protocol will vary depending upon various factorsincluding the age, body weight, general health, sex and diet of thepatient; the determination of specific administration procedures wouldbe routine to an ordinarily skilled artisan.

[0259] To be effective therapeutically as central nervous systemtargets, the compounds should readily penetrate the blood-brain barrierwhen peripherally administered. Compounds that cannot penetrate theblood-brain barrier can be effectively administered by anintraventricular route or by other methods recognized in the art. See,for example, U.S. Pat. Nos. 5,846,565; 5,651,986; and 5,626,862,incorporated herein by reference.

Dosage

[0260] The compounds and compositions of the present invention may beadministered by a single dose, multiple discrete doses, or continuousinfusion. The compounds are well suited to continuous infusion. Pumpmeans, particularly subcutaneous pump means, are preferred forcontinuous infusion.

[0261] Dose levels on the order of about 0.001 to about 10,000 mg/kg ofthe active ingredient compound are useful in the treatment of the aboveconditions, with preferred levels being about 0.1 to about 1,000 mg/kg,and more preferred levels being about 1 to about 100 mg/kg. The specificdose level for any particular patient will vary depending upon a varietyof factors, including the activity and the possible toxicity of thespecific compound employed; the age, body weight, general health, sex,and diet of the patient; the time of administration; the rate ofexcretion; drug combination; the severity of the particular diseasebeing treated; and the form of administration. Typically, in vitrodosage-effect results provide useful guidance on the proper doses forpatient administration. Studies in animal models are also helpful. Theconsiderations for determining the proper dose levels are well known inthe art.

Aministration Regimen

[0262] For the methods of the present invention, any administrationregimen well known to an ordinarily skilled artisan for regulating thetiming and sequence of drug delivery can be used and repeated asnecessary to effect treatment. Such regimen may include pretreatmentand/or co-administration with additional therapeutic agents.

Co-Administration with Other Treatments

[0263] The compounds and compositions of the present invention may beused alone or in combination with one or more additional agent(s) forsimultaneous, separate or sequential use.

[0264] The additional agent(s) may be any therapeutic agent(s) known toan ordinarily skilled artisan, including without limitation: one or morecompound(s) of the invention; steroids, for example, hydrocortisonessuch as methylprednisolone; anti-inflammatory or anti-immune drugs, suchas methotrexate, azathioprine, cyclophosphamide or cyclosporin A;interferon-β; antibodies, such as anti-CD4 antibodies; agents which canreduce the risk of a second ischemic event, such as ticlopidine;chemotherapeutic agents; immunotherapeutic compositions; electromagneticradiosensitizers; and morphine.

[0265] The compounds of the present invention can be co-administeredwith one or more therapeutic agents either (i) together in a singleformulation, or (ii) separately in individual formulations designed foroptimal release rates of their respective active agent. Each formulationmay contain from about 0.01% to about 99.99% by weight, preferably fromabout 3.5% to about 60% by weight, of a compound of the presentinvention, as well as one or more pharmaceutical excipients, such aswetting, emulsifying, and pH buffering agents.

Preparations of Compounds

[0266] The compounds of the present invention can be readily prepared bystandard techniques of organic chemistry, utilizing the generalsynthetic pathways depicted below in Schemes I, II, III, and IV.Precursor compounds are either commercially available or may be preparedby methods known to a person of skill in the art.

EXAMPLES

[0267] The following examples are illustrative of the present inventionand are not intended to be limitations thereon. Unless otherwiseindicated, all percentages are based upon 100% by weight of the finalcomposition.

Example 1 Procedure for the Synthesis of3-(2-Mercaptoethyl)-[1,1′-Biphenyl]-2,3′-Dicarboxylic Acid (10) (SchemeI) 3-(2,2-Dimethyl-4-oxo-4H-1,3-benzodioxin-5-yl)-benzoic acid, ethylester

[0268] To a solution of2,2-dimethyl-5-trifluoromethane-sulfonyloxy-4H-1,3-benzodioxin-4-one(2.0 g, 5.8 mmol), 3-ethoxycarbonylphenylboronic acid (1.34 g, 6.9 mmol)and anhydrous K₂CO₃ powder (2.61 g, 18.9 mmol) in DMF (30 mL) was addedtetrakis(triphenylphosphine)palladium (0.202 g, 0.175 mmol). The mixturewas heated at reflux for 2 h. The reaction mixture was allowed to coolto room temperature (“rt”) and 1 N HCl (25 mL) was added. The mixturewas extracted with EtOAc (3×25 mL). The combined extracts were washedwith water and brine, then dried over MgSO₄, filtered, and concentrated.The crude material was purified by flash chromatography (1:15EtOAc/hexanes) to afford3-(2,2-dimethyl4-oxo4H-1,3-benzodioxin-5-yl)-benzoic acid, ethyl ester(1.2 g, 63%) as a white solid: ¹H NMR (CDCl₃) δ1.39 (t, J=7.1 Hz, 3H),1.80 (s, 6H), 4.39 (q, J=7.0 Hz, 2H), 7.01 (d, J=8.0 Hz, 2H), 7.47-7.57(m, 3H), 8.00 (t, J=1.5 Hz, 1H) 8.07 (dt, J=7.5, 1.5 Hz, 1H).

3-Hydroxy-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethyl ester

[0269] To a solution of3-(2,2-dimethyl-4-oxo4H-1,3-benzodioxin-5-yl)-benzoic acid, ethyl ester(1.4 g, 4.3 mmol) in methanol (10 mL) was added sodium methoxide (0.5 Min methanol, 25 mL) at 0° C. The solution was stirred at rt for 15 min.The reaction was quenched by addition of 1 N HCl (30 mL) and extractedwith EtOAc (3×30 mL). The combined organic extracts were dried overMgSO₄ and concentrated to afford3-hydroxy-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethyl ester (1.2 g,95%) as a yellow solid: ¹H NMR (CDCl₃) δ3.43 (s, 3H), 3.93 (s, 3H), 6.79(dd, J=7.5, 0.9 Hz, 1H) 7.04 (dd, J=7.5, 0.9 Hz, 1H), 7.43 (m, 3H), 7.93(m, 1H), 8.02 (dm, J=7.0 Hz, 1H), 10.8 (s, 1H).

3-Trifluoromethanesulfonyloxy-[1,1′-biphenyl]-2,3′-dicarboxylic acid,dimethyl ester

[0270] To a solution of 3-hydroxy-[1,1′-biphenyl]-2,3′-dicarboxylicacid, dimethyl ester (1.1 g, 3.8 mmol) in dichloromethane (15 mL) wereadded pyridine (1.00 mL, 12.3 mmol) and trifluromethanesulfonicanhydride (0.90 mL, 5.4 mmol) at 0° C. The solution was stirred at 0° C.for 2 h. Aqueous 1 N HCl (20 mL) was added, and the mixture wasextracted with dichloromethane (3×20 mL). The combined organic extractswere washed with water and brine, dried over MgSO₄, filtered, andconcentrated to give3-trifluoromethanesulfonyloxy-[1,1′-biphenyl]-2,3′-dicarboxylic acid,dimethyl ester (1.4 g, 87%) as a yellow solid: ¹H NMR (CDCl₃) 67 3.72(s, 3H), 3.94 (s, 3H), 7.38-7.62 (m, 5H), 8.08 (m, 2H).

3-Ethenyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethyl ester

[0271] A mixture of3-trifluoromethanesulfonyloxy-[1,1′-biphenyl]-2,3′-dicarboxylic acid,dimethyl ester (1.3 g, 3.1 mmol), tetrakis(triphenylphosphine)palladium(0.36 g, 0.31 mmol), LiCl (0.94 g, 22.2 mmol), triethylamine (0.6 mL,4.3 mmol) and tri-n-butyl(vinyl)tin (1.0 mL, 3.4 mmol) in 1,4-dioxane(30 mL) was heated at reflux under N₂ for 4 h. After cooling to rt, themixture was filtered through a plug of silica gel and the filtrate wasconcentrated. Purification by flash chromatography (1:10 EtOAc/hexanes)provided 3-ethenyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethylester (0.91 g, 99%) as a white solid: ¹H NMR (CDCl₃) δ3.61 (s, 3H), 3.92(s, 3H), 5.40 (d, J=11.1 Hz, 1H), 5.79 (d, J=17.5 Hz, 1H), 6.87 (dd,J=17.4, 11.0 Hz, 1H), 7.31 (d, J=7.5 Hz, 1H), 7.44 7.49 (m, 2H), 7.56(dm, J=7.5 Hz, 1H), 7.62 (d, J=8.0 Hz, 1H), 8.03 (dm, J=1H), 8.08 (t, 1,J=1.5 Hz, 1H).

3-[2-(Acetylthio)ethyl]-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethylester

[0272] To a solution of 3-ethenyl-[1,1′-biphenyl]-2,3′-dicarboxylicacid, dimethyl ester (0.85 g, 2.9 mmol) in benzene (10 mL) was addedthioacetic acid (2.1 mL, 29.4 mmol) followed by AIBN (0.053 g, 0.32mmol). The solution was deoxygenated for 30 min by bubbling nitrogenthrough the solution and then heated at reflux for 4 h. Saturatedaqueous NaHCO₃ (20 mL) was added to the solution and the mixture wasextracted with EtOAc (2×20 mL). The combined organic extracts werewashed with water and brine, dried over MgSO₄, filtered, andconcentrated. The residue was purified by flash chromatography (1:12EtOAc/hexanes) to give3-[2-(acetylthio)ethyl]-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethylester (0.51 g, 48%) as an off white solid: ¹H NMR (CDCl₃) δ2.35 (s, 3H),2.93 (m, 2H), 3.14 (m, 2H), 3.62 (s, 3H), 3.93 (s, 3H), 7.29 (dd, J=7.6,0.9 Hz, 1H), 7,35 (dd, J=7.5, 0.8 Hz, 1H), 7.44 (d, J=7.6 Hz, 1H), 7.48(d, J=7.5 Hz, 1), 7.55 (dt, J=8.0, 1.5 Hz, 1H), 8.03 (dt, J=7.9, 1.5 Hz,1H), 8.07 (t, J=1.5 Hz, 1H).

3-(2-Mercaptoethyl)-[1,1′-biphenyl]-2,3′-dicarboxylic acid, 2-methylester

[0273] To a deoxygenated solution of3-[2-(acetylthio)ethyl]-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethylester (0.50 g, 1.34 mmol) in THF (3.5 mL) was added a deoxygenatedsolution of NaOH (0.38 g, 9.4 mmol) in water (3.5 mL). The mixture wasstirred overnight, and 1 N HCI (20 mL) was added. The mixture wasextracted with EtOAc (3×20 mL). The combined organic extracts werewashed with water and brine, dried over MgSO₄, filtered, andconcentrated to afford3-(2-mercaptoethyl)-[1,1′-biphenyl]-2,3′-dicarboxylic acid, 2-methylester (0.35 g, 83%) as an off white solid: ¹H NMR (CDCl₃) δ1.46 (t,J=8.0 Hz, 1H), 2.83 (m, 2H), 3.00 (m, 2H), 3.60 (s, 3H), 7.33-7.31 (m,2H), 7.46 (t, J=7.7 Hz, 1H), 7.52 (t, J=7.7 Hz, 1H), 7.61 (dm, J=7.9 Hz,1H), 8.10 (dm, J=7.9 Hz, 1H), 8.14 (m, 1H).

3-(2-Mercaptoethyl)-[1,1′-biphenyl]-2,3′-dicarboxylic acid (10)

[0274] To a deoxygenated suspension of sodium ethanethiolate (0.135 g,1.60 mmol) in DMF (0.5 mL) was added a solution of3-(2-mercaptoethyl)-[1,1′-biphenyl]-2,3′-dicarboxylic acid, 2-methylester (0.10 g, 0.32 mmol) in DMF (0.5 mL). Argon was bubbled through themixture for 10 min. The reaction was heated at 100° C. for 1 h and 200°C. for another hour. After the mixture cooled to rt, the reaction wasquenched with 1 N HCl (20 mL) and was extracted with EtOAc (3×20 mL).The combined organic extracts were washed with water and brine, driedover MgSO₄, filtered, and concentrated to afford3-(2-mercaptoethyl)-[1,1′-biphenyl]-2,3′-dicarboxylic acid (0.055 g,57%) as a white solid: ¹H NMR (CDCl₃) δ1.51 (t, J=8.0 Hz, 1H), 2.87-2.93(m, 2H), 3.12-3.08 (m, 2H), 7.37 (m, 2H), 7.57-7.47 (m, 2H), 7.70 (dm,J=7.9 Hz, 1H), 7.98 (dm, J=7.8 Hz, 1H), 8.30 (m, 1H); ¹³C NMR (CDCl₃)δ26.2, 38.8, 128.1, 129.3, 129.7, 129.8, 129.9 (2C), 130.5, 133.3,134.4, 137.7, 139.1, 141.2, 172.3, 176.9. Anal. Calcd for C₁₆H₁₄O₄S: C,63.56; H, 4.67; S, 10.61. Found: C, 63.65; H, 4.88; S, 10.33.

Example 2 Procedure for the Synthesis of2-[(4-Carboxyphenyl)Methoxyl]-6-(2-Mercaptoethyl)-Benzoic Acid (7)(Scheme II) 5-Ethenyl-2,2-dimethyl-4H-1,3-benzodioxin-4-one

[0275] A mixture of2,2-dimethyl-5-trifluoromethanesulfonyloxy-4H-1,3-benzodioxin4-one (9.90g, 30.3 mmol), tributyl(vinyl)tin (10.10 g, 31.9 mmol), lithium chloride(8.70 g, 205 mmol), and triethylamine (5.0 mL, 36.0 mmol) in 1,4-dioxane(300 mL) was deoxygenated by bubbling nitrogen through the mixture for 1h. To the mixture was added tetrakis(triphenylphosphine)palladium (3.40g, 2.90 mmol) and the mixture was heated at 100° C. for 3 h. The mixturewas allowed to cool to rt and was filtered. The filtrate wasconcentrated and purified by flash chromatography (1:12, EtOAc/hexanes)to provide 5-ethenyl-2,2-dimethyl-4H-1,3-benzodioxin-4-one (5.00 g, 81%)as a yellow oil: ¹H NMR: (CDCl₃) δ1.72 (s, 6H), 5.43 (dd, J=11.0, 1.3Hz, 1), 5.72 (dd, J=17.5, 1.3 Hz, 1 Hz, 1H), 6.89 (d, J=8.0 Hz, 1H),7.27 (d, J=8.0 Hz, 1H), 7.47 (t, J=8.0 Hz, 1H), 7.73 (dd, J=17.5, 11.0Hz, 1H).

2-Ethenyl-6-hydroxybenzoic acid, methyl ester

[0276] To 5-ethenyl-2,2-dimethyl-4H-1,3-benzodioxin-4-one (4.01 g, 19.6mmol) was added 0.5 M sodium methoxide in methanol (85 mL, 42.5 mmol) atrt. Aqueous 1 N HCl (100 mL) was added to the solution after 15 min. Thecloudy solution was extracted with ether (2×100 mL). The combinedorganic extracts were washed with H₂O (50 mL) and brine (50 mL), driedover Na₂SO₄, filtered, and concentrated to afford2-ethenyl-6-hydroxybenzoic acid, methyl ester (2.0 g, 57%) as a yellowoil. This material was used without further purification in the nextstep: ¹H NMR (CDCl₃) δ3.96 (s, 3H), 5.26 (dd, J=10.8, 1.5 Hz, 1H), 5.49(dd, J=17.3, 1.5 Hz, 1H), 6.95 (m, 2H), 7.23-7.39 (m, 2H), 11.12 (s,1H).

2-Ethenyl-6-[4-(methoxycarbonyl)phenyl]methoxy-benzoic acid, methylester

[0277] To a stirred solution of the above material (0.500 g, 2.8 mmol)in acetone (10 mL) were added K₂CO₃ (1.50 g, 10.9 mmol) and methyl4-(bromomethyl)benzoate (0.71 g, 3.10 mmol) at rt. The mixture wasstirred under nitrogen for 3 h and filtered. The filtrate wasconcentrated and residue was purified by flash chromatography (1:10EtOAc/hexanes) to provide2-ethenyl-6-[4-(methoxycarbonyl)phenyl]methoxy-benzoic acid, methylester (0.73 g, 80%) as a white solid: ¹H NMR (CDCl₃) δ3.92 (s, 6H), 5.17(s, 2H), 5.37 (dd, J=11.1, 1.0 Hz, 1H), 5.78 (dd, J=17.6, 0.9 Hz, 1H),6.70 (dd, 1, J=17.4, 11.1 Hz, 1H), 6.83 (d, J=7.8 Hz, 1H), 7.20 (d,J=8.0 Hz, 1H) 7.29 (t, J=80 Hz, 1H), 7.46 (d, J=8.4 Hz, 2H), 8.04 (d,J=8.3 Hz, 2H).

2-[2-(Acetylthio)ethyl]-6-[4-(methoxycarbonyl)phenyl]methoxy-benzoicacid, methyl ester

[0278] To a solution of2-ethenyl-6-[4-(methoxycarbonyl)phenyl]methoxy-benzoic acid, methylester (0.71 g, 2.18 mmol) in benzene (10 mL) was added thioacetic acid(1.80 mL, 25.2 mmol) followed by AIBN (37 mg, 0.23 mmol). After nitrogenwas bubbled through the solution for 30 min, the solution was heated atreflux for 4 h. The reaction was allowed to cool to rt and saturatedNaHCO₃ (20 mL) was added. The mixture was extracted with EtOAc (3×20mL). The combined organic extracts were washed with water and brine,dried over MgSO₄, filtered and concentrated in vacuo. The residue waspurified by flash chromatography (1:10 EtOAc/hexanes) to give2-[2-(acetylthio)ethyl]-6-[4-(methoxycarbonyl)phenyl]methoxy-benzoicacid, methyl ester (0.50 g, 60%) as a clear oil: ¹H NMR (CDCl₃) δ2.34(s, 3H), 2.85-2.82 (m, 2H), 3.07-3.10 (m, 2H), 3.92 (s, 3H), 3.94 (s,3H), 5.16 (s, 2H), 6.81 (d, J=8.2 Hz, 1H), 6.92 (d, J=7.7 Hz, 1H), 7.28(t, J=8.2 Hz, 1H), 7.46 (d, J=8.3 Hz, 2H), 8.04 (d, J=8.3 Hz, 2H).

2-[(4-Carboxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid (7)

[0279] To a deoxygenated solution of2-[2-(acetylthio)ethyl]-6-[4-(methoxycarbonyl)phenyl]methoxy-benzoicacid, methyl ester (0.20 g, 0.50 mmol) in 95% EtOH (3 mL) was added adeoxygenated solution of KOH (0.463 g, 8.3 mmol) in 95% EtOH (3 mL)under nitrogen. The solution was heated at reflux overnight and quenchedby addition of 1 N HCl (20 mL). The mixture was extracted with EtOAc(3×20 mL) and the combined organic extracts were washed with water andbrine, then dried over MgSO₄, filtered, and concentrated. Purificationby flash chromatography (1:1 dichloromathane/hexanes with 1% aceticacid) provided 2-[(4-carboxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoicacid (0.077 g, 46%) as a white solid: ¹H NMR (CD₃OD) δ2.75 (m, 2H), 2.92(m, 2H), 5.22 (s, 2H), 6.93 (d, J=7.5 Hz, 1H), 6.98 (d, J=8.2 Hz, 1H),7.30 (t, J=8.3 Hz, 1H), 7.55 (d, J=7.9 Hz, 2H), 8.02 (d, J=8.0 Hz, 2H);¹³C NMR (CD₃OD) δ26.5, 39.8, 71.1, 112.4, 123.9, 126.9, 128.3, 131.3,131.7, 131.8, 139.9, 144.2, 156.7, 170.0, 172.3. Anal. Calcd forC₁₇H₁₆O₅S: C, 61.43; H, 4.85; S, 9.65. Found: C, 61.16; H, 4.95; S,9.44.

Example 3 Procedure for the Synthesis of4-Mercaptomethyl-[1,1′-Biphenyl]-2,3′-Dicarboxylic Acid (26) (SchemeIII) 4-Methyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid

[0280] To a solution of 2-bromo-5-methylbenzoic acid (5.00 g, 23.3 mmol)in DME (100 mL) were added 3-carboxyphenylboronic acid (3.86 g; 23.3mmol), a solution of Na₂CO₃ (9.90 g, 93 mmol) in H₂O andtetrakis(triphenylphosphine)palladium. The mixture was stirred at 90° C.for 4 days. The mixture was allowed to cool to rt, diluted with EtOAc(50 mL), and washed with a saturated NaHCO₃ solution. The aqueous layerwas separated, acidified with 10% HCl, and extracted with EtOAc (3×20mL). The combined extracts were dried over MgSO₄ and concentrated. Thecrude material was purified by column chromatography (9:1 hexanes/EtOAc1% acetic acid) to afford 4-methyl-[1,1′-biphenyl]-2,3′-dicarboxylicacid (2.20 g, 37%) as a solid: ¹H NMR (DMSO-d₆) δ2.40 (s, 3H), 7.30 (m,1H), 7.42 (m, 1H), 7.52-7.57 (m, 2H), 7.60 (s, 1H), 7.85 (s, 1H),7.91-7.92 (m, 1H).

4-Methyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethyl ester

[0281] To a solution of 4-methyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid(2.20 g, 8.6 mmol) in methanol (150 mL) was added conc. H₂SO₄ (1.6 mL)and the mixture was heated at reflux overnight. The solvent was removedunder a reduced pressure and the residue was partitioned betweensaturated aqueous NaHCO₃ solution and EtOAc (20 mL). The organic layerwas dried over MgSO₄ and concentrated to give4-methyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethyl ester (2.26 g,92%) as a crude material. This product was used for the next reactionwithout further purification: ¹H NMR (DMSO-d₆) δ2.41 (s, 3H), 3.58 (s,3H), 3.88 (s, 3H), 7.36-7.38 (m, 1H), 7.47-7.48 (m, 1H), 7.56-7.58 (m,2H), 7.62 (s, 1H), 7.82 (s, 1H), 7.94-7.96 (m, 1H).

4-Bromomethyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethyl ester

[0282] To a solution of 4-methyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid,dimethyl ester (2.26 g, 7.9 mmol) in CCl₄ (50 mL) were added benzoylperoxide (0.010 g, 0.04 mmol) and NBS (1.42 g, 8.0 mmol), and themixture was refluxed for 3 days. The reaction mixture was allowed tocool to rt, filtered, and concentrated. The residue was purified bycolumn chromatography (95:5 to 90:10 hexanes/EtOAc) to afford4-bromomethyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethyl ester(1.71 g, 60%): ¹H NMR (DMSO-d₆) δ3.61(s, 3H), 3.88 (s, 3H), 4.84 (s,2H), 7.48-7.50 (d, J=8.0 Hz, 1H), 7.59-7.60 (m, 2H), 7.72-7.75 (m, 1H),7.85 (s, 1H), 7.90 (m, 1H), 7.97-7.99 (m, 1H).

4-Acetylthiomethyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethylester

[0283] To a solution of 4-bromomethyl-[1,1′-biphenyl]-2,3′-dicarboxylicacid, dimethyl ester (1.59 g, 4.4 mmol) in acetone (75 mL) was addedpotassium thioacetate (0.60 g, 5.3 mmol), and the mixture was refluxedfor 1 h. The mixture was allowed to cool to rt, filtered, andconcentrated. The residual product was purified by column chromatography(hexanes/EtOAc, 9/1) to afford4-acetylthiomethyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethylester (1.21 g, 76%): ¹H NMR (DMSO-d₆) δ2.39 (s, 3H), 3.60 (s, 3H), 3.88(s, 3H), 4.23 (s, 2H), 7.42-7.44 (d, J=8.0 Hz, 1H), 7.57-7.60 (m, 3H),7.74 (s 1H), 7.83 (s, 1H), 7.96-7.99 (m, 1H).

4-Mercaptomethyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid (26)

[0284] To a solution of4-acetylthiomethyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid, dimethylester (0.27 g, 0.75 mmol) in deoxygenated THF was added a degassedsolution of sodium hydroxide (0.12 g, 3.0 mmol) in H₂O (5 mL) at rt.After 24 h, additional solution of sodium hydroxide (0.09 g) in H₂O (2mL) was added to the reaction mixture and the mixture was stirred for 24h. The mixture was acidified with 10% HCl and extracted with EtOAc. Theextract was dried over MgSO₄ and concentrated. The crude material waspurified by column chromatography with (9:1 dichloromethane/EtOAc with1% acetic acid) to afford4-mercaptomethyl-[1,1′-biphenyl]-2,3′-dicarboxylic acid (0.20 g, 92%) asa white solid: ¹H NMR (DMSO-d₆) δ3.10 (t, J=8.03 Hz, 1H), 3.89 (d,J=8.03 Hz, 2H), 7.43 (d, J=7.53 Hz, 1H), 7.58-7.65 (m, 3H), 7.83 (d,J=2.01 Hz, 1H), 7.92 (s, 1H), 7.97-8.00 (m, 1H). Anal. Calcd. forC₁₅H₁₂O₄S.0.5AcOH: C, 60.37; H, 4.43; O, 25.13; S, 10.07. Found: C,60.28; H, 4.45; S, 10.15.

Example 4 Procedure for the Synthesis of2-Carboxymethyl-6-(2-Mercaptoethyl)-Benzoic Acid (48) (Scheme IV)2-Hydroxy-6-methoxycarbonylmethyl-benzoic acid methyl ester

[0285] To a solution of 2-carboxymethyl-6-hydroxy-benzoic acid (5.021 g,25.6 mmol) in DMF (100 mL) at 0° C. were added K₂CO₃l (3.567 g, 25.9mmol) and CH₃l (7.9 mL, 51.9 mmol). After stirring under nitrogen at 0°C. for 4 h, the reaction was partitioned between H₂O (100 mL) and ether(150 mL). The aqueous layer was acidified with 1 N HCl and extractedwith EtOAc (200 mL). The EtOAc layer was dried over MgSO₄ andconcentrated in vacuo to give the crude2-hydroxy-6-methoxycarbonylmethyl-benzoic acid methyl ester as acolorless oil (3.2 g, 56%): ¹H NMR (CDCl₃) δ3.69 (s, 3H), 3.89 (s, 5H),6.72 (d, J=6.7 Hz, 1H), 6.96 (d, J=8.0 Hz, 1H), 7.33-7.38 (m, 1H).

2-Methoxycarbonylmethyl-6-trifluoromethanesulfonyloxy-benzoic acidmethyl ester

[0286] To a solution of 2-hydroxy-6-methoxycarbonylmethyl-benzoic acidmethyl ester (2.51 g, 11.2 mmol) in CH₂Cl₂ (50 mL) at 0° C. were addedtriflic anhydride (3.0 mL, 17.8 mmol) and pyridine (2.40 mL, 29.7 mmol).The solution was allowed to warm to rt overnight and was concentrated invacuo. The residue was diluted with EtOAc (100 mL), washed with 1 N HCI(25 mL), saturated aqueous NaHCO₃ (25 mL), H₂O (25 mL), and brine (25mL). The organic layer was dried over MgSO₄ and concentrated. Theresidue was purified by flash chromatography (10% EtOAc/hexanes) toprovide 2-methoxycarbonylmethyl-6-trifluoromethanesulfonyloxy-benzoicacid methyl ester as an oil (3.5 g, 90%): ¹H NMR (CDCl₃) δ3.86 (s, 3H),4.06 (s, 2H), 4.10 (s, 3H), 7.4 (d, J=7.6 Hz, 1H), 7.50 (d, J=7.6 Hz,1H), 7.67 (t, J=8.2 Hz, 1H).

2-Methoxycarbonylmethyl-6-vinyl-benzoic acid methyl ester

[0287] A mixture of2-methoxycarbonylmethyl-6-trifluoromethanesulfonyloxy-benzoic acidmethyl ester (2.705 g, 7.6 mmol), LiCl (2.355 g, 55.6 mmol), NEt₃ (1.5mL, 10.8 mmol), Pd[P(C₆H₅)₃]₄(1.09 g, 0.94 mmol), and tributyl(vinyl)tin (2.85 mL, 9.75 mmol) in dioxane (50 mL) was heated at reflux.After 3 h, the reaction was allowed to cool to rt, filtered through apad of silica gel and concentrated. The crude product was purified byflash chromatography on SiO₂(13% EtOAc/hexanes) to afford2-methoxycarbonylmethyl-6-vinyl-benzoic acid methyl ester as a yellowsolid (1.50 g, 84%): ¹H NMR (CDCl₃) δ3.67 (s, 3H), 3.89 (s, 5H), 5.32(d, J=10.9 Hz, 1H), 5.68 (d, J=17.4 Hz, 1H), 6.82 (dd, J=17.4, 10.9 Hz,1H), 7.20 (d, J=7.1 Hz, 1H), 7.36 (m, 1H), 7.50 (d, J=7.8 Hz, 1H).

2-(2-Acetylsulfanyl-ethyl)-6-methoxycarbonylmethyl-benzoic acid methylester

[0288] Nitrogen was bubbled through a solution of2-methoxycarbonylmethyl-6-vinyl-benzoic acid methyl ester (1.31 g, 5.60mmol), thioacetic acid (4.00 mL, 56.0 mmol), and AIBN (0.184 g, 1.10mmol) in benzene (30 mL) for 3 h at rt to remove oxygen. The mixture washeated at reflux for 3 h. The solution was allowed to cool to rt andsaturated aqueous NaHCO₃ (100 mL) was added. The mixture was extractedwith EtOAc (100 mL) and the organic layer was washed with H₂O (100 mL)and brine (100 mL), then dried over MgSO₄ and concentrated in vacuo.Flash chromatography on SiO₂ (10% EtOAc/hexanes) provided2-(2-acetylsulfanyl-ethyl)-6-methoxycarbonylmethyl-benzoic acid methylester as a yellow solid (0.555 g, 33%): ¹H NMR (CDCl₃): δ2.40 (s, 3H),2.94-2.99 (m, 2H), 3.13-3.18 (m, 2H), 3.75 (s, 3H), 3.78 (s, 2H), 3.99(s, 3H), 7.25 (d, J=7.6 Hz, 1H), 7.32 (d, J=7.6 Hz, 1H), 7.42 (t, J=7.6Hz, 1H).

2-Carboxymethyl-6-(2-mercapto-ethyl)-benzoic acid (48)

[0289] A deoxygenated mixture of2-(2-acetylsulfanyl-ethyl)-6-methoxycarbonylmethyl-benzoic acid methylester (0.555 g, 1.77 mmol) and 6 N KOH (3 mL) in EtOH (7 mL) was heatedat reflux for 18 h. After allowing to cool to rt, the mixture waspartitioned between 1 N HCl (75 mL) and EtOAc (100 mL). The organiclayer was washed with H₂O (100 mL), brine (100 mL), dried over MgSO₄ andconcentrated in vacuo. The crude product was dissolved in ether (100mL), filtered to remove some unidentified solid material, andconcentrated to give 2-carboxymethyl-6-(2-mercapto-ethyl)-benzoic acidas a white solid (0.310 g, 73%): ¹H NMR (CDCl₃) δ1.37 (t, J=8.2 Hz, 1H),2.76 (m, 2H), 3.06 (m, 2H), 3.90 (s, 2H), 7.10 (d, J=7.6 Hz, 1H), 7.21(d, J=6.9 Hz, 1H), 7.34 (t, J=7.8 Hz, 1H): ¹³C NMR (CDCl₃) δ27.25,40.43, 41.83, 131.58, 131.64, 132.45, 132.55, 134.66, 141.41, 176.14,179.40. Anal. Calcd for C, H₁₁H₁₂O₄S: C, 54.99; H, 5.03; S, 13.35.Found: C, 55.20; H, 5.26; S, 13.32.

Example 5A In Vitro Inhibition of NAALADase Activity

[0290] Various compound of the invention were tested for in vitroinhibition of NAALADase activity, and the results are provided below inTable II. TABLE II Compound No. IC₅₀ 1 613 2 9170 3 71.5 4 380 5 215 684.5 7 89.8 8 11.5 9 28.8 10 84.8 11 188 12 2580 13 266 14 160 15 23.716 4.28 17 7 18 10.5 19 2.65 20 18 21 1150 26 24.3 27 85 28 3200 29 605030 3780 31 100000 32 25 33 95 34 0.9 35 1.2 36 100 37 70 38 0.8 39 40 401 41 1250 42 300 43 104 44 954 45 35200 46 12200 47 75400 48 43

Protocol for Assaying In Vitro Inhibition of NAALADase Activity

[0291] The following were combined in assay tubes: 100 μL of 10 mMCoCl₂, 250 μL of 200 mM Tris chloride, 100 μL tissue, 100 μL of 10 mMNAALADase inhibitor in Bakers H₂O, and Bakers H₂O to make a total volumeof 950 μL. Each assay tube was then incubated for 10 minutes in a 37° C.water bath. 50 μL of 3-H-NAAG was then added to each assay tube andincubated for an additional 15 minutes in a 37° C. water bath. The assaywas stopped by adding 1.0 ml of 0.1 M sodium phosphate.

[0292] Glutamate released by the action of the NAALADase enzyme wasseparated from the assay solution using an anion exchange resin. Theresin was equilibrated to 25° C., 2.0 ml of the resin was added to aPasteur pipette pre-loaded with a single glass bead, and each column waswashed twice with distilled H₂O. A column was placed over ascintillation vial and 200 μL of an assay sample was loaded onto thecolumn. After draining, glutamate was eluted using two 1.0 ml washes of1 M formic acid. After addition of 10 ml of scintillation cocktail, eachsample was counted for 2 minutes on a scintillation counter.

Example 5B In Vitro Assay on Ischemia

[0293] To examine the in vitro effect of the compounds of the inventionon ischemia, cortical cell cultures were treated with various compoundsof the invention during an ischemic insult utilizing potassium cyanideand 2-deoxyglucose, and for one hour thereafter. For a description ofthe experimental method used, see Vornov et al., J. Neurochem., Vol. 65,No. 4, pp. 1681-1691 (1995). The results are provided below in TableIII. Neuroprotective effect is expressed as EC₅₀, the concentration ofthe compound, which is required to cause a 50% reduction in glutamatetoxicity following an ischemic insult. TABLE III Compound No. EC₅₀ 1 8463 10000 4 9600 6 84 7 96 8 29 9 19 25 2000 26 151 27 728 29 2400 30 619

Example 6 Effect of NAALADase Inhibition on TGF-β in In Vitro IschemiaModel

[0294] Compound C was added to ischemia cell cultures to determine itseffect on TGF-β levels during stroke. The experimental data, set forthin FIGS. 1 and 2, show increased concentrations of TGF-β1 and TGF-β2(FIG. 2) in ischemic cell cultures treated with Compound C. The resultsindicate that NAALADase inhibition promotes the release of endogenousTGF-β's from glial cells, which in turn provides neuroprotection forneighboring neurons.

[0295] TGF-β neutralizing antibodies were then added to the ischemiccell cultures. FIG. 3 shows that the TGF-β neutralizing antibodiesblocked the neuroprotective effect of Compound C in the in vitroischemia model. By contrast, FIG. 4 shows that the addition of anothergrowth factor antibody, FGF antibody, did not block the neuroprotectiveeffect of Compound C. The results indicate that NAALADase inhibitionspecifically affects TGF-β levels during stroke.

Example 7

[0296] Effect of NAALADase Inhibition on TGF-β in In Vivo Ischemia Model

[0297] The effect of TGF-β neutralizing antibodies on theneuroprotective effect of Compound C was also studied in rats followingMCAO. FIG. 6 shows that treatment of MCAO rats with Compound C caused asignificant rise in TGF-β1 levels during both occlusion and reperfusion,as assessed by microdialysis. The results indicate that NAALADaseinhibition provides neuroprotection, at least in part, by regulatingendogenous TGF-β's.

[0298] Additionally, FIG. 5 shows that TGF-β neutralizing antibodiessignificantly attenuated the neuroprotective effect of Compound C invivo. One of ordinary skill in the art can appreciate that theregulation of TGF-β's by NAALADase inhibitors may have implications notonly in stroke, but also in other diseases, disorders and conditionsincluding, without limitation, neurological diseases, psychiatricdiseases, demyelinating diseases, prostate cancer, inflammation,diabetes and angiogenesis.

Example 8 In Vivo Assay of NAALADase Inhibitors on Neuropathic Pain inSTZ Model

[0299] Male Sprague-Dawley rats (200-225 g) were rendered diabetic byintravenous administration of streptozotocin (“STZ”, 70 mg/kg inphosphate buffered saline). Diabetic animals were divided into fivegroups: one group receiving Compound A (10 mg/kg or 1 mg/kg), Compound D(10 mg/kg or 1 mg/kg) or vehicle. Another group of animals (non-STZtreated) served as non-diabetic controls. Drug/vehicle treatment wasstarted in diabetic animals 45 days post-STZ administration. STZ-induceddiabetic rats were tested for sensitivity to a heat source as soon asblood glucose levels rose to 320 mg/dl or above (30 days post STZ). Therats were then acclimated to a Hargreaves apparatus and thermalnociception was monitored using an infrared heat source directed intothe dorsal surface of the hindpaw, and the time taken for the animal toremove its paw noted to the nearest 0.1 seconds (see Hargreaves et al.,J. Biol. Chem. (1988) 263(36):19392-7 for a description of theexperimental method). The intensity of the beam source was adjusted suchthat the mean latency for control animals (non-STZ treated) wasapproximately 10 seconds. Each animal was tested 8 times and the meandifference score (between mean non-diabetic control latency and meandiabetic latency) are graphically presented in FIGS. 7A and 7B. Diabeticrats displayed a hyperalgesia (shorter response latency) compared tonon-diabetic controls, starting 30 days post STZ treatment andprogressively worsening in vehicle treated rats. This hyperalgesicresponse was completely reversed in diabetic rats receiving treatmentwith Compound D or A (10 mg/kg i.p. daily). Thus, the results show thatNAALADase inhibition attenuates neuropathic pain.

Example 9 In Vivo Assay of NAALADase Inhibitors on Neuropathic Pain inCCI Model

[0300] Sciatic nerve ligation, consisting of 4 ligatures tied looselyaround the sciatic nerve at 1 mm intervals proximal to the nervetrifurcation, was performed on rats. Following sciatic nerve ligation,the rats exhibited a thermal hyperalgesia and allodynia. The rats werehabituated to a Hargreaves apparatus. An infrared heat source wasdirected onto the dorsal surface of each rat's hindpaws and the timetaken for the rat to withdraw its paws was noted. The difference inscores between the latency of the response for the paw on the operatedside versus the paw on the unoperated control side was determined.

Compound C

[0301] The rats received either Compound C (50 mg/kg i.p. daily) or avehicle starting 10 days post surgery. Treatment with Compound Cdramatically normalized the difference scores between the two pawscompared to the continued hyperalgesic vehicle treated controls. Normal(unoperated) rats had approximately equal latencies for both paws. Thiseffect was significant starting at 11 days of drug treatment andpersisted through to the end of the study (for 21 days of daily dosing).The difference scores are graphically presented in FIG. 8. The resultsshow that NAALADase inhibition attenuates CCI-associated hyperalgesia.

Compound 9

[0302] The rats were treated with either Compound 9 (10, 1 or 0.1 mg/kg)or a vehicle for 15 days after sciatic nerve ligation. Thermal painresponses were measured at days 0, 1, 5, 8, 12 and 15. The differencesin scores for the rats treated with a vehicle and the rats treated withCompound 9 are graphically presented in FIG. 13. The results show thattreatment with Compound 9 normalized the difference in scores betweenthe operated and unoperated paws compared to the continued hyperalgesicvehicle-treated rats.

Compound 10

[0303] The rats were treated with either Compound 10 (0.1 mg/kg) or avehicle for 18 days after sciatic nerve ligation. Thermal pain responseswere measured at days 0, 1, 5, 12, and 18. The differences in scores forthe rats treated with a vehicle and the rats treated with Compound 10are graphically presented in FIG. 14. The results show that treatmentwith Compound 10 normalized the difference in scores between theoperated and unoperated paws compared to the continued hyperalgesicvehicle-treated rats.

Example 10 In Vivo Assay of NAALADase Inhibitors on Progression ofNeuropathic Pain in BB/W Models

[0304] Male BB/W rats (BRI, Mass) spontaneously develop a cell mediatedautoimmune destruction of pancreatic B cells, resulting in onset ofinsulin-dependent (Type I) diabetes (Awata, Guberski, Endocrinology(1995) 136(12):5731-5). These rats have been characterized and shown todemonstrate neuropathies with accompanying neural deficits such as fiberloss and degeneration, changes which are correlative with those seen inperipheral nerve of human diabetic patients (Yagihasi, J. Peripher.Nerv. Syst. (1997) 2(2):113-32). This renders them valuable forexperimental trials of new compounds for future treatments of this majordisorder. In the present study, Compound A and Compound D were examinedfor their ability to alter the progression of diabetic neuropathy. Therats received daily injection of Compound A or Compound D (10 mg/kgi.p.) or vehicle, starting at the onset of diabetes (hyperglycemia) andup to 6 months thereafter. Another group of non-diabetic rats alsoreceiving vehicle were tested. All animals were continuously monitoredfor body weight, urine volume, blood sugar and glycated haemoglobin. Inthe first month of the study, all animals were tested for thermalnociception in a Hargreaves apparatus, weekly. After the first monththis was done biweekly and then monthly. The testing consists ofdirecting an infrared heat source onto the dorsal surface of the rathindpaw and noting the time taken for the animal to remove its paw (seeHargreaves et al., J. Biol. Chem. (1988) 263(36):19392-7 for adescription of the experimental method). Each animal was tested 8 timesand the mean withdrawal latency noted.

[0305] The results are graphically presented in FIG. 11. The resultsshow that diabetic rats displayed a hyperalgesia (shorter responselatency) compared to non-diabetic controls. Diabetic drug-treated rats(both Compound A and Compound D) displayed longer withdrawal latenciesthan diabetic vehicle-treated rats, starting after 4 weeks of treatmentand persisting through the six months of treatment.

[0306] Nerve conduction velocity was also measured every two weeksthrough the first eight weeks of treatment and every month thereafterthrough to the six months of treatment (see De Koning et al., Peptides,Vol. 8, No. 3, pp. 415-22 (1987) for a description of the experimentalmethod). The results are graphically presented in FIG. 12. Diabeticanimals generally showed a reduction in nerve conduction velocitycompared to non-diabetic controls. However, diabetic animals receivingdaily injections of NAALADase inhibitor (either Compound A or Compound Dat a dose of 10 mg/kg) showed significantly less severe nerve conductiondeficits than did the diabetic controls receiving vehicle treatment.This was apparent starting at 8 weeks of treatment and persisted to asimilar degree through to the six month termination point of the study.Diabetic vehicles, on the other hand, showed a progressive deteriorationin nerve conduction velocity from 6 to 16 weeks after start of vehicleadministration which was maintained through to six months.

Example 11 In Vivo Assay of NAALADase Inhibitors on Diabetic Neuropathyin STZ Model

[0307] Motor and sensory nerve conduction velocity was also measured inSTZ-diabetic animals after 4, 8 and 12 weeks of treatment (see De Koninget al., supra, for a description of the experimental method). Briefly,stimulating needle electrodes were inserted close to the sciatic andtibial nerves with recording electrodes being placed subcutaneously overthe distal foot muscles, in anesthetized rats. The results aregraphically presented in FIGS. 9A, 9B, 10A and 10B. Diabetic animalsreceiving vehicle showed a significant reduction in both motor andsensory nerve conduction compared to non-diabetic animals. Treatmentwith 10 mg/kg of Compound A daily for 4, 8 and 12 weeks all tended toimprove (increase) both motor and sensory nerve conduction velocities,with a significant improvement being observed after 12 weeks and 8 weeksfor motor and sensory nerve conduction velocity, respectively (FIGS. 9Aand 9B). The lower dose of Compound A tested (1 mg/kg) had similareffects. Treatment of animals with Compound D at either dose alsoincreased both motor and sensory nerve conduction velocities above thatof diabetic controls, significantly so after 12 weeks of treatment forthe 10 mg/kg treatment group (FIGS. 10A and 10B) and at the earlier timeperiods after treatment with the 1 mg/kg dose. Thus, the results showthat NAALADase inhibition alters the progression of diabetic neuropathy.

Example 11A In Vivo Assay of NAALADase Inhibitors—Reversal of DiabeticNeuropathy in STZ Model

[0308] General Method for STZ Model—Delayed Dosing

[0309] Rats (200-225 grams) were injected with STZ (70 mg/kg) into thetail vein. Diabetes (>350 mg/dl) was confirmed in all rats, 4 weeksafter STZ administration. Rats were left untreated until 35-49 daysafter STZ. Compound D (1, 3, or 10 mg/kg), Compound E (10 mg/kg), orvehicle were dosed daily p.o. following confirmation of hyperalgesiaand/or nerve conduction velocity deficits. In separate experiments,onset of treatment was delayed until 60 to 90 days after SZadministration. Nerve conduction velocity or withdrawal response tothermal stimulation of hind paws was measured at intervals, usuallybi-weekly for thermal response and monthly for nerve conductionvelocity.

[0310] General Method for db/db Mice Study

[0311] Spontaneously diabetic mice (db/db mice) and non-diabeticlittennates were obtained from Jackson labs. Mice were left untreateduntil 7-8 months of age (or after 4-5 months of chronic diabetes) andthen dosed daily with compound F (1 mg/kg) p.o. Nerve conductionvelocity was measured prior to the onset and after eight weeks oftreatment.

[0312] Nerve Conduction Velocity Measurements

[0313] Sensory and motor nerve conduction velocities were evaluatedusing the method of De Koning and Gispen (Peptides 8: 415-422, 1987).Electrophysiological evaluation was carried out within one hour ofdosing. Animals were anesthetized with isoflurane and stimulating needleelectrodes were inserted close to the sciatic nerve at the sciatic notchand the tibial nerve near the ankle. Recording electrodes were placedover the foot muscles. Stimuli were applied and responses recorded.Motor and sensory nerve conduction velocities were calculated bymeasuring the distance between the sciatic notch and ankle sites, andthe latency between the M-wave and the H-reflex.

[0314] Thermal Hyperalgesia

[0315] Animals were acclimated to the apparatus for at least 5 min. Aninfra-red source was placed under below the plantar surface of the rathind-paw. The intensity of the source was adjusted so that latency fornormal rats was about 10 secs. Animals were tested for thermal responselatency according to the method of Hargreaves et al (Pain 77-88, 1988).Each animal was tested 8 times (4 each hind limb) and the latency ofresponse recorded automatically to nearest 0.1 sec. An average of thelast 4 measurements for each paw was calculated (8 total measurements)and noted for each rat.

[0316]FIG. 22 shows the effect of NAALADase inhibitor (Compound D andCompound E) treatment on neuropathic pain abnormalities in STZ diabeticrats. All rats showed apparent hyperalgesia compared to non-diabeticsprior to NAALADase inhibitor treatment (5 weeks post STZ). However,within two weeks of treatment neuropathic hyperalgesia was reversedtowards normal in both NAALADase inhibitor treated groups. This reversalpersisted throughout the subsequent hypoalgesic phase usually seen inprolonged diabetic-STZ rats, with a reduced hypoalgesic phase displayedin NAALADase treated rats.

[0317]FIG. 23 shows the motor nerve conduction velocity measurements inSTZ diabetic rats and non-diabetic controls prior to and at time periodsafter NAALADase inhibitor treatment. Within 8 weeks of dosing, bothNAALADase inhibitors Compound D and Compound E reversed the motor nerveconduction velocity towards normal (non-diabetic values). This effectpersisted through 12 weeks of treatment.

[0318]FIG. 24 depicts sensory nerve conduction velocity deficits,similarly tested. NAALADAse inhibitor treatment similarly reversedsensory nerve conduction velocity deficits, significantly so after only2 weeks of treatment.

[0319]FIG. 25 shows neuropathic pain abnormalities in another experimentwhere lower doses (1 and 3 mg/kg) of the NAALADase inhibitor Compound Dinitiated after 7 weeks of STZ treatment were tested. Significantreduction in pain abnormalities were again apparent with both doses ofCompound D.

[0320]FIGS. 26 and 27 show sensory and motor nerve conduction velocityrespectively in these chronically diabetic STZ rats treated with thelower doses of Compound D. Sensory nerve conduction was significantlyimproved towards normal within 4 weeks of treatment whereas motor nerveconduction remained unimproved by these low doses, even 8 weeks afterdosing.

[0321]FIGS. 28 and 29 show sensory and motor nerve conduction velocitymeasurements generated from an external CRO in a similar chronicallydiabetic STZ model, where rats were left untreated until 60 days afterSTZ treatment. Partial reversal of both deficits was again produced byCompound D treatment. FIG. 30 shows the same where treatment was delayedyet further, until 90 days after STZ.

[0322]FIG. 31 shows nerve conduction velocity measurements from agenetic mouse model of diabetes, at 6-7 months of age (after about 4months of chronic diabetes). A significant impairment in sensory NCV wasapparent at this time. FIG. 32 shows the nerve conduction velocity inthese mice after 8 weeks of treatment with another, more potentNAALADase inhibitor administered at 1 mg/kg daily. Significantimprovement in the sensory nerve conduction was apparent following drugtreatment.

Example 12 Effect of NAALADase Inhibitors on Onset of ALS

[0323] The effect of NAALADase inhibitors on the onset of ALS was testedusing the transgenic mice model of familial amyotrophic lateralsclerosis (“FALS”), which is detailed in Gurney, M., Annals of Neurology(1996) 39:147-157, and otherwise well known in the art. One month oldtransgenic G1H mice were treated with daily intraperitoneal injectionsof a vehicle (50 mM HEPES-buffered saline) or a NAALADase inhibitor (50mg/kg Compound A). Clinical symptoms of the mice were monitored daily.The onset of clinical disease was scored by examining each mouse for itsshaking of limbs when suspended in the air by its tail, cross spread ofspinal reflexes, hindlimb paralysis, body weight and wheel runningactivity.

[0324] The results, set forth below in Table IV, show that disease onsetwas delayed in mice treated with a NAALADase inhibitor. TABLE IV EffectOf Naaladase Inhibitor On Onset Of Clinical Disease DISEASE ONSETDISEASE ONSET COMPOUND A VEHICLE STUDY (days) (days) DIFFERENCE Study 1221 189 32 Study 2 166 141 25

Example 13 Effect of NAALADase Inhibitor on ALS Survival and ClinicalSymptoms

[0325] The effect of NAALADase inhibitors on ALS survival and clinicalsymptoms was tested using again the transgenic mice model of FALS. Onemonth old transgenic G1H mice were treated daily with a vehicle (50 mMHEPES-buffered saline) or Compound B p.o. (by oral administration).Clinical symptoms of the mice were monitored twice a week. Such symptomsincluded shaking of limbs, gait, dragging of hind limbs, crossing oflimbs, righting reflex and mortality. Gait and crossing of limbs weregraded on an arbitrary scale ranging from 0 to 3, with 0 representingmost normal and 3 representing least normal, e.g. severest difficulty inwalking or crossing limbs. Righting reflex was measured by the time(seconds) it took the mice to right themselves when placed on theirsides on a flat surface.

[0326] The results, set forth in FIGS. 15-21, show that survival wasprolonged and clinical symptoms were attenuated in mice treated with aNAALADase inhibitor.

Example 14 Protective Effect of NAALADase Inhibitors in Experimental RatGlaucoma

[0327] Experimental Protocol

[0328] All experiments complied with the Association for Research inVision and Ophthalmology Statement for the Use of Animals in Ophthalmicand Vision Research. 82 male Brown Norway rats (Rattus norvegicus), eachweighing approximately 250 gm, were treated using procedures approved bythe Animal Care Committee of the Johns Hopkins University School ofMedicine. The rats were housed with a 12 hour light/12 hour dark cycleand fed ad libitum.

[0329] EXPERIMENTAL GLAUCOMA: Unilateral elevation of intraocularpressure (“IOP”) was produced in 56 rats by microinjection of hypertonicsaline into episcleral veins, following procedures described inMorrison, J. et al., IOVS (March 1998) 39:526-531. Beginning on the dayof IOP elevation, the rats were treated daily with intraperitonealinjections of either a vehicle (23 rats with 50 mM HEPES-bufferedsaline) or a NAALADase inhibitor (11 rats with 10 mg/kg of Compound Aand 22 rats with 10 mg/kg of Compound B). 11 saline treated rats, 11Compound A treated rats and 11 Compound B treated rats were sacrificedat 8 weeks, and the remaining rats at 12 weeks, after initial IOPelevation.

[0330] OPTIC NERVE TRANSECTION: The optic nerve was transectedunilaterally in 26 rats under intraperitoneal pentobarbital anesthesia.The conjunctiva was opened with scissors and the optic nerve exposed bytraction on extraocular muscles. The transection was performed withmicroscissors 5 mm posterior to the globe, with specific attention toavoidance of injury to major ocular blood vessels. Immediately aftertransection, the retina was examined ophthalmoscopically to assure thatthe retinal arterial blood supply was not disrupted. The conjunctiva wasclosed with absorbable suture and the eye dressed with antibioticointment. Beginning on the day of transection, the rats were treateddaily with intraperitoneal injections of either a vehicle (9 rats with50 mM HEPES-buffered saline) or a NAALADase inhibitor (8 rats with 10mg/kg of Compound A and 9 rats with 10 mg/kg of Compound B). 5 salinetreated rats, 3 Compound A treated rats, and 4 Compound B treated ratswere sacrificed at 2 weeks, and the remaining rats at 4 weeks, aftertransection.

[0331] OPTIC NERVE COUNTING: The rats were sacrificed by exsanguinationunder deep pentobarbital anesthesia. They were perfused through theheart with 2% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphatebuffer, pH 7.2, and the eyes with attached optic nerves were removed. Across-section of the optic nerve from both experimental (glaucoma ortransection) and control eyes was removed 1.5 mm posterior to the globe,1 mm in thickness, and post-fixed in 2% osmium tetroxide in buffer.These were processed into epoxy resin, sectioned at 1 micron and stainedwith toluidine blue.

[0332] The area of the optic nerve cross-section was measured byoutlining its outer border at 10×magnification on an image analysissystem (Universal Imaging Corp., Westchester, Pa.) with Synsys digitalcamera and Metamorph software. Three area measurements were taken andthe mean value was determined. To measure the density and fiber diameterdistributions, images were captured with a 100×phase contrast objectivefrom 10 different areas of each nerve. These were edited to eliminatenon-neural objects and the size of each axon internal to the myelinsheath (its minimum diameter) and the density of axons/square mm werecalculated for each image and nerve. The mean density was multiplied bytotal nerve area to yield fiber number for each nerve. The total fibernumber in glaucoma or transection nerves was compared to the normal,fellow eye of each rat to yield a percent loss value. The number ofaxons counted among the 10 images was an approximately 20% sample of the80-90,000 axons in normal rat nerves. The person measuring axon numberwas masked to the protocol conducted on the nerves.

[0333] Results

[0334] EXPERIMENTAL GLAUCOMA: The mean fiber percent difference in thesaline-treated, control rats was significantly lower in their glaucomaeyes compared to their normal eyes, with a mean fiber loss of14.44±5.75% (n=11 rats; Table V) in the 8 week follow-up group, and8.15±7.84% in the 12 week follow-up group (n=12 rats; Table VI).

[0335] By contrast, there was no significant loss of fibers in eitherthe 8 week or 12 week NAALADase inhibitor-treated rats. The mean percentfiber loss in each NAALADase inhibitor-treated group was statisticallyless than the loss in the saline-treated, control groups (at 8 weeks,p=0.05 for Compound A and p=0.02 for Compound B). TABLE V ExperimentalGlaucoma Results 8 WEEK IOP INTEGRAL FIBER PERCENT GROUP NDIFFERENCE^(a) NUMBER DIFFERENCE^(b) Compound 11  85 ± 37.5 79156 ±2436* −1.82 ± 2.92 A Compound 11 116 ± 33.2 80785 ± −0.82 ± 2.97 B2121** Control 11 104 ± 26.4 68295 ± 4617 14.44 ± 5.75

[0336] TABLE VI Experimental Glaucoma Results 12 WEEK IOP INTEGRAL FIBERPERCENT GROUP N DIFFERENCE^(a) NUMBER DIFFERENCE^(b) Compound B 11 109 ±45.2 90504 ± 1718 −3.21 ± 2.86 Control 12 158 ± 66.5 79827 ± 6783   8.15± 7.84

[0337] Differences in IOP Integral Difference are not significant(p>0.05). Differences in Percent Difference between drug-treated andsaline-treated, control rats at 8 weeks post insult are significant(p=0.05* and p=0.02**).

[0338] OPTIC NERVE TRANSECTION: The experimental transection datasuggest a slowing or rescue of ultimate RGC death in rats treated withNAALADase inhibitors at 2 weeks after transection. At 2 weeks aftertransection, both drug-treated groups had more remaining RGC axons thandid the saline-treated, control group, judged either by absolute numberof fibers or percent difference between transected eye and normal eye ineach rat (Table VII). Rats treated with Compound A and Compound B had,respectively, 3 times and twice as many remaining axons as thesaline-treated rats. All or nearly all RGC die within the first 2 monthsafter transection, regardless of any pharmacological treatment. Thus, by4 weeks after transection, more than 80% of RGC axons were gone in allgroups (Table VII). At 4 weeks after transection, there were nosignificant differences between the drug-treated rats and thesaline-treated rats. TABLE VII Optic Nerve Transection PERCENT 2 WEEKSSURVIVAL N FIBER NUMBER DIFFERENCE^(a) Compound A 3 26,426 ± 23,025 65.3± 30.9 Compound B 4 19,550 ± 11,383 75.3 ± 14.8 Control 5  8,220 ± 9,33790.2 ± 10.7

[0339] TABLE VIII Optic Nerve Transection PERCENT 4 WEEKS SURVIVAL NFIBER NUMBER DIFFERENCE^(a) Compound A 5 13,599 ± 7,868 82.4 ± 8.9Compound B 5  5,162 ± 5,017 93.4 ± 6.2 Control 4 10,449 ± 8,157 86.9 ±10.6

[0340] Differences in Percent Difference between drug-treated andsaline-treated, control rats are not statistically significant (p=0.05).

Example 15 Neuroprotective Effect of NAALADase Inhibitors in TransgenicMouse Model of Huntington's Disease

[0341] Behavioral Testing (Rotarod)

[0342] Transgenic HD mice of the N171-82Q strain and non-transgeniclittermates were treated with NAALADase inhibitor Compound B (30 mg/kg)or a vehicle from 10 weeks of age. The mice were placed on a rotatingrod (“rotarod”). The length of time at which the mouse fell off therotarod was recorded as a measure of motor coordination. FIG. 33 showsthat transgenic HD mice treated with Compound B stayed longer on therotarod than similar transgenic HD mice treated with a vehicle. Thetreatment with Compound B had no effect on the rotarod performance ofnormal non-HD mice.

[0343] The total distance traveled by the mice was also recorded as ameasure of overall locomotion. FIG. 34 shows that while the vehicletreated HD mice demonstrated the lowest mean locomotor score, thetreatment with NAALADase inhibitor had no apparent effect on overalllocomotion.

[0344] Survival

[0345] The effects of Compound B and vehicle on the survival oftransgenic HD mice (N171-82Q) were evaluated. Thirteen mice (six maleand seven female) were assigned to the Compound B treatment group, andfourteen mice (six male and eight female) were assigned to the vehicletreatment group. Treatment was continued until all the mice died.

[0346]FIG. 35 shows the survival distributions over time by treatmentgroup. The median survival time is 184 days for the Compound B treatmentgroup, and 158.5 days for the vehicle treatment group. Although theCompound B treatment group had a longer median survival time than thevehicle treatment group, the difference is not statistically significant(p-value=0.07).

[0347]FIGS. 36 and 37 show the survival distributions over time bytreatment group and sex. When analyzing the results specific to sex,female mice treated with Compound B had significantly prolonged survivaltime (p-value=0.03) compared to their vehicle treated counterparts.Within the vehicle treatment group, the males have better survival timesthan the females although this trend was not observed in the Compound Btreatment group. The data suggest that sex may influence survivaldistributions over time.

Example 16

[0348] A patient is suffering from any disease, disorder, or conditionwhere NAALADase levels are altered, including any of the diseases,disorders, or conditions described above. The patient may then beadministered an effective amount of a compound of the invention. It isexpected that after such treatment, the patient would not suffer anysignificant injury due to, would be protected from further injury dueto, or would recover from the disease, disorder or condition.

[0349] All publications, patents and patent applications identifiedabove are herein incorporated by reference, as though set forth hereinin full.

[0350] The invention being thus described, it will be apparent to thoseskilled in the art that the same may be varied in many ways withoutdeparting from the spirit and scope of the invention. Such variationsare included within the scope of the following claims.

We claim:
 1. A compound of formula Ia or a pharmaceutically acceptableequivalent:

wherein: R¹, R², R³, and R⁴ are independently hydrogen or C₁-C₃ alkyl;and A¹, A², A³, and A⁴ are independently hydrogen, C₁-C₉ alkyl, C₂-C₉alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle, heterocycle, halo,hydroxy, sulfhydryl, nitro, amino, cyano, isocyano, thiocyano,isothiocyano, formamido, thioformamido, sulfo, sulfino, C₁-C₉alkylsulfonyl, C₁-C₉ alkoxy, C₂-C₉ alkenoxy, phenoxy, or benzyloxy,wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle,heterocycle, alkoxy, alkenoxy, phenoxy, and benzyloxy are independentlyunsubstituted or substituted with one or more substituent(s).
 2. Acompound according to claim 1 wherein: R¹, R², R³, R⁴, A², A³, and A⁴are hydrogen; and A¹ is hydrogen, —(CH₂)_(n)—W, or —Y—(CH₂)_(n)—W,wherein: n is 0-3; Y is O, S, or NR wherein R is hydrogen or C1-C4alkyl; and W is C1-C6 alkyl or phenyl, wherein W is unsubstituted orsubstituted with C1-C4 alkyl, C1-C4 alkoxy, carboxy, or halo.
 3. Acompound according to claim 2, wherein the compound is an enantiomer orpart of an enantiomer-enriched mixture.
 4. A compound according to claim2 wherein: A¹ is —(CH₂)_(n)—W, or —Y—(CH₂)_(n—W,) n is 1 or 2, and Y isO.
 5. A compound according to claim 4 wherein the compound is:2-(2-mercaptoethyl)-benzoic acid;5-[(4-carboxyphenyl)methoxy]-2-(2-mercaptoethyl)-benzoic acid;2-(2-mercaptoethyl)-5-(phenylmethoxy)-benzoic acid;2-(carboxymethoxy)-6-(2-mercaptoethyl)-benzoic acid;5-[(3-carboxyphenyl)methoxy]-2-(2-mercaptoethyl)-benzoic acid;2-(2-mercaptoethyl)-6-(phenylmethoxy)-benzoic acid;2-[(2-carboxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;2-[(4-carboxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;3-(2-mercaptoethyl)-[1,1′-biphenyl]-2,3′-dicarboxylic acid;2-(3,3-dimethylbutoxy)-6-(2-mercaptoethyl)-benzoic acid;2-(2-mercaptoethyl)-6-(2-phenylethoxy)-benzoic acid;2-[(2-chlorophenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;2-[[3-carboxy-5-(1,1-dimethylethyl)phenyl]methoxy]-6-(2-mercaptoethyl)-benzoicacid; 3-(2-mercaptoethyl)-[1,1′-biphenyl]-2,4′-dicarboxylic acid;2-[(4-carboxy-2-methoxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;2-[(4-carboxy-3-methoxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;2-[(2-bromo-4-carboxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;2-[(3-bromo-4-carboxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;2-(2-mercaptoethyl)-6-phenoxy-benzoic acid;2-(2-mercaptoethyl)-6-phenylamino-benzoic acid;2-(2-mercaptoethyl)-6-(phenylthio)-benzoic acid;5′-(1,1-dimethylethyl)-3-(2-mercaptoethyl)-[1,1′-biphenyl]-2,3′-dicarboxylicacid; 2-[(4-chlorophenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;2-(biphenyl-2-ylmethoxy)-6-(2-mercaptoethyl)-benzoic acid;2-[(3-bromo-5-carboxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;2-[(2-bromo-5-carboxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;2-(2-mercaptoethyl)-6-[(4-methoxyphenyl)methoxy]-benzoic acid;2-(2-mercaptoethyl)-6-[(4-methylphenyl)methoxy]-benzoic acid;2-[(4-bromo-3-carboxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;2-[(2-carboxy-5-methoxyphenyl)methoxy]-6-(2-mercaptoethyl)-benzoic acid;2-(3-carboxy-benzyloxy)-6-(2-mercapto-ethyl)-benzoic acid;2-(4-bromo-benzyloxy)-6-(2-mercapto-ethyl)-benzoic acid;2-(4-tert-butyl-benzyloxy)-6-(2-mercapto-ethyl)-benzoic acid;2-(3-bromo-benzyloxy)-6-(2-mercapto-ethyl)-benzoic acid;2-(2-mercapto-ethyl)-6-methoxy-benzoic acid;2-(3-chloro-benzyloxy)-6-(2-mercapto-ethyl)-benzoic acid;3-(2-mercapto-ethyl)-biphenyl-2-carboxylic acid; or2-carboxymethyl-6-(2-mercapto-ethyl)-benzoic acid; or a pharmaceuticallyacceptable salt thereof.
 6. A compound according to claim 5 wherein thecompound is: 2-(2-mercaptoethyl)-6-(phenylmethoxy)-benzoic acid;3-(2-mercaptoethyl)-[1,1′-biphenyl]-2,3′-dicarboxylic acid; or2-carboxymethyl-6-(2-mercapto-ethyl)-benzoic acid; or a pharmaceuticallyacceptable salt thereof.
 7. A method for inhibiting NAALADase enzymeactivity, treating a glutamate abnormality, effecting a neuronalactivity, treating a prostate disease, treating cancer, inhibitingangiogenesis or effecting a TGF-β activity, comprising administering toa mammal in need of such inhibition, treatment or effect, an effectiveamount of a compound of claim
 1. 8. The method of claim 7, wherein saidmethod is for treating a glutamate abnormality selected from the groupconsisting of a compulsive disorder, stroke, demyelinating disease,schizophrenia, Parkinson's disease, amyotrophic lateral sclerosis,anxiety, anxiety disorder, memory impairment and glaucoma.
 9. The methodof claim 7, wherein said method is for effecting a neuronal activityselected from the group consisting of stimulation of damaged neurons,promotion of neuronal regeneration, prevention of neurodegeneration andtreatment of a neurological disorder.
 10. The method of claim 9, whereinthe neuronal activity is treatment of a neurological disorder and saidneurological disorder is pain, diabetic neuropathy, peripheralneuropathy caused by physical injury or disease state, traumatic braininjury, physical damage to spinal cord, stroke associated with braindamage, a demyelinating disease or a neurological disorder relating toneurodegeneration.
 11. The method of claim 7, wherein said method is fortreating cancer.
 12. The method of claim 7, wherein said method is forinhibiting angiogenesis.
 13. The method of claim 7, wherein said methodis for treating a TGF-ρ abnormality selected from the group consistingof neurodegenerative disorder, extra-cellular matrix formation disorder,cell-growth related disease, infectious disease, immune related disease,epithelial tissue scarring, collagen vascular disease,fibroproliferative disorder, connective tissue disorder, inflammation,inflammatory disease, respiratory distress syndrome, infertility, anddiabetes.
 14. A method for detecting a disease, disorder or conditionwhere NAALADase levels are altered, comprising: (i) contacting a sampleof bodily tissue or fluid with a compound according to claim 1, whereinsaid compound binds to any NAALADase in said sample; and (ii) measuringthe amount of any NAALADase bound to said sample, wherein the amount ofNAALADase is diagnostic for said disease, disorder, or condition.
 15. Amethod for detecting a disease, disorder or condition where NAALADaselevels are altered, comprising: (i) labeling a compound according toclaim 1, with an imaging reagent; (ii) administering to an animal ormammal an effective amount of the labeled compound; (iii) allowing saidlabeled compound to localize and bind to NAALADase present in saidanimal or mammal; and (iv) measuring the amount of NAALADase bound tosaid labeled compound, wherein the amount of NAALADase is diagnostic forsaid disease, disorder, or condition.
 16. A diagnostic kit for detectinga disease, disorder, or condition where NAALADase levels are altered,comprising a compound according to claim 1 labeled with a marker.
 17. Apharmaceutical composition comprising: (i) an effective amount of acompound according to claim 1; and (ii) a pharmaceutically acceptablecarrier.
 18. A compound of formula Ib or a pharmaceutically acceptableequivalent:

wherein A¹, A², A³ and A⁴ are independently hydrogen, C₁-C₉ alkyl, C₂-C₉alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle, heterocycle, halo,hydroxy, sulfhydryl, nitro, amino, cyano, isocyano, thiocyano,isothiocyano, formamido, thioformamido, sulfo, sulfino, C₁-C₉alkylsulfonyl, C₁-C₉ alkoxy, C₂-C₉ alkenoxy, phenoxy, or benzyloxy,wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle,heterocycle, alkoxy, alkenoxy, phenoxy, and benzyloxy are independentlyunsubstituted or substituted with one or more substituent(s), wherein ifA¹ is chloro, fluoro, amino, or thiomethyl then A², A³, and A⁴ may notall be hydrogen, and wherein at least one of A¹, A², A³, and A⁴ is nothydrogen.
 19. A compound according to claim 18 wherein: A², A³, and A⁴are hydrogen; and A¹ is —(CH₂)_(n)—Ar or —Y—(CH₂)_(n)—Ar, wherein n is0-3, Y is O, S, or NR wherein R is hydrogen or C₁-C₄ alkyl, and Ar isphenyl, unsubstituted or substituted with C₁-C₄ alkyl, carboxy, or halo.20. A compound according to claim 19, wherein the compound is anenantiomer or part of an enantiomer-enriched mixture.
 21. A compoundaccording to claim 19 wherein the compound is:5-(mercaptomethyl)-2-(2-phenylethoxy)-benzoic acid;4-(mercaptomethyl)-[1,1′-biphenyl]-2,3′-dicarboxylic acid;5-(mercaptomethyl)-2-(phenylmethoxy)-benzoic acid; or4-bromo-3-(mercaptomethyl)-benzoic acid; or a pharmaceuticallyacceptable salt thereof.
 22. A compound according to claim 21 whereinthe compound is 4-(mercaptomethyl)-[1,1′-biphenyl]-2,3′-dicarboxylicacid or a pharmaceutically acceptable salt thereof.
 23. A method forinhibiting NAALADase enzyme activity, treating a glutamate abnormality,effecting a neuronal activity, treating a prostate disease, treatingcancer, inhibiting angiogenesis or effecting a TGF-β activity,comprising administering to a mammal in need of such inhibition,treatment or effect, an effective amount of a compound of claim 18 24.The method of claim 23, wherein said method is for treating a glutamateabnormality selected from the group consisting of a compulsive disorder,stroke, demyelinating disease, schizophrenia, Parkinson's disease,amyotrophic lateral sclerosis, anxiety, anxiety disorder, memoryimpairment and glaucoma.
 25. The method of claim 23, wherein said methodis for effecting a neuronal activity selected from the group consistingof stimulation of damaged neurons, promotion of neuronal regeneration,prevention of neurodegeneration and treatment of a neurologicaldisorder.
 26. The method of claim 25, wherein the neuronal activity istreatment of a neurological disorder and said neurological disorder ispain, diabetic neuropathy, peripheral neuropathy caused by physicalinjury or disease state, traumatic brain injury, physical damage tospinal cord, stroke associated with brain damage, a demyelinatingdisease or a neurological disorder relating to neurodegeneration. 27.The method of claim 23, wherein said method is for treating cancer. 28.The method of claim 23, wherein said method is for inhibitingangiogenesis.
 29. The method of claim 23, wherein said method is fortreating a TGF-β abnormality selected from the group consisting ofneurodegenerative disorder, extra-cellular matrix formation disorder,cell-growth related disease, infectious disease, immune related disease,epithelial tissue scarring, collagen vascular disease,fibroproliferative disorder, connective tissue disorder, inflammation,inflammatory disease, respiratory, distress syndrome, infertility, anddiabetes.
 30. A method for detecting a disease, disorder or conditionwhere NAALADase levels are altered, comprising: (i) contacting a sampleof bodily tissue or fluid with a compound according to claim 18, whereinsaid compound binds to any NAALADase in said sample; and (ii) measuringthe amount of any NAALADase bound to said sample, wherein the amount ofNAALADase is diagnostic for said disease, disorder, or condition.
 31. Amethod for detecting a disease, disorder or condition where NAALADaselevels are altered, comprising: (i) labeling a compound according toclaim 18 with an imaging reagent; (ii) administering to an animal ormammal an effective amount of the labeled compound; (iii) allowing saidlabeled compound to localize and bind to NAALADase present in saidanimal or mammal; and (iv) measuring the amount of NAALADase bound tosaid labeled compound, wherein the amount of NAALADase is diagnostic forsaid disease, disorder, or condition.
 32. A diagnostic kit for detectinga disease, disorder, or condition where NAALADase levels are altered,comprising a compound according to claim 18 labeled with a marker.
 33. Apharmaceutical composition comprising: (i) an effective amount of acompound according to claim 18; and (ii) a pharmaceutically acceptablecarrier.
 34. A compound of formula I or a pharmaceutically acceptableequivalent:

or a pharmaceutically acceptable equivalent, wherein: X is—(CR¹R²)_(n)SH, —O(CR¹R²)₂SH, —S(CR¹R²)₂SH, or —NR(CR¹R²)₂SH; n is 1-3;and R, R¹, R², A¹, A², A³ and A⁴ are independently hydrogen, C₁-C₉alkyl, C₂-C₉ alkenyl, C₂-C₉ alkynyl, aryl, heteroaryl, carbocycle,heterocycle, halo, hydroxy, sulfhydryl, nitro, amino, cyano, isocyano,thiocyano, isothiocyano, formamido, thioformamido, sulfo, sulfino, C₁-C₉alkylsulfonyl, C₁-C₉ alkoxy, C₂-C₉ alkenoxy, phenoxy, or benzyloxy,wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle,heterocycle, alkoxy, alkenoxy, phenoxy, and benzyloxy are independentlyunsubstituted or substituted with one or more substituent(s).